Materials

All chemicals were from Sigma-Aldrich (Oakville, ON, Canada), and used

without any additional purification. All organic solvents were from Caledon labs

(Georgetown, ON, Canada). BcMag DEAE magnetic beads were from Bioclone Inc. (San

Diego, CA). Desalting columns (illustra NAP-5) were from GE Life Sciences (Quebec,

Canada). Amicon Ultra-0.5 centrifugal filters were from Fisher Scientific (Ontario,

Canada). CdSxSe1-x/ZnS (core/shell) alloyed semiconductor nanoparticles with emission

wavelengths of 525 nm, 575 nm and 630 nm were from Cytodiagnostics Inc. (Burlington,

ON, Canada).

Peptide sequences were from CanPeptide Inc (Montreal, QC, Canada):

(6-Maleimidohexanoic acid) – G(Aib)GHHHHHH

DNA sequences were from IDT DNA (Coralville, IA, USA):

Probe DNA 5’- Thiol – TTT TTT TTT TCT TAC TTC CAT GAT TTC TTT AAC TAT GCC G– 3’

Displacer DNA (Proximal)

5’ – AAA GAA ATC ATG GAA GTA AGT TTT TTT TTT – Thiol-3’

Displacer DNA (Distal)

5’ – Thiol – TTT TTT TTT TCG GCA TAG TTA AAG AAA TCA-3’

Target DNA 5’ – CCA CGG TGA TAT CGT CCA CCC AGG TGT TCG GCG TGG TGT AGA GCA TTA CGC TGC GAT GGA TCC CGG CAT AGT TAA AGA AAT CAT GGA AGT AAG – 3’

Buffers:

Borate buffer: 100 mM borate, pH 9.25

Tris-borate buffer (TB): 100 mM tris, 100 mM borate, pH 7.3

Phosphate buffered saline (PBS): 10 mM phosphate, 2.7 mM potassium chloride, 137

mM sodium chloride, pH 7.4

Tris-borate EDTA buffer (TBE): 89 mM Tris, 89 mM boric acid, 2 mM

ethylenediaminetetraacetic acid, pH 8.3

Instrumentation

UV-visible spectra were obtained using a HP8452A diode-array

spectrophotometer (Hewlett Packard Corporation, Palo Alto, CA, USA). The agarose gels

were imaged using a BioRad ChemiDoc XRS+ (Bio-Rad, Hercules, CA, USA).

TEM/SEM images were obtained using a Hitachi S-5200 electron microscope (Hitachi

High Technologies America, Pleasanton, CA, USA). Fluorescence spectra was obtained

using one of two instruments: (1) a PTI QuantaMaster spectrofluorimeter equipped with a

xenon arc lamp (Ushio, Cypress, CA) as the excitation source and a red-sensitive R928P

photomultiplier tube (Hamamatsu, Bridgewater, NJ) as the detector, and (2) a Nikon

Eclipse L150 epifluorescence microscope, equipped with a 25 mW diode laser (λ = 402

nm; Radius 402, Coherent Inc., Santa Clara, CA) as the excitation source and a diode

array spectrometer (QE65000, Ocean Optics Inc., Dunedin, FL) as the detector. The laser

radiation was passed through a filter cube with a ZET 405/20x excitation filter and a

Z405rdc dichroic mirror (Chroma Technologies Corp., Bellow Falls, VT) followed by a

40x Nikon ELWD Plan Fluor Objective (Numerical Aperture: 0.60) objective lens. Time-

resolved fluorescence decay measurements were obtained using a dye laser that was

pumped using a pulsed N2 laser. DPS (4,4’ – (1,2-ethenediyl)bis-1,1’-biphenyl) dissolved

in dioxane was used in the dye laser, with the emission tuned to 402 nm.

Methods

Synthesis of gold nanoparticles

6 nm gold nanoparticles (6AuNP)

These nanoparticles were synthesized using the procedure of Slot et al.

{JW:1985wa}Trisodium citrate (34 μmol) and tannic acid (2.9 μmol) was dissolved in 20

mL of deionized water. In a separate flask, gold (III) chloride (33 μmol) was dissolved in

80 mL of water. Both solutions were heated to 60 oC, and then mixed together with

vigorous stirring. Once the solution turned red, it was heated to 95 oC and then cooled in

an ice bath.

13 nm gold nanoparticles (13AuNP)

The protocol described by Liu et al. {Liu:2006hd}was followed to obtain 13 nm gold

nanoparticles. Gold (III) chloride (0.1 mmol) was added to 100 mL of water, which was

then brought to a rolling boil under vigorous stirring. Trisodium citrate (0.39 mmol),

dissolved in 10 mL of water, was added to the boiling solution. The solution was

continuously heated and stirred for 20 min, after it which it was allowed to cool to room

temperature.

30 nm gold nanoparticles (40AuNP)

The protocol proposed by Perrault et al.{Perrault:2009ua} was used for the synthesis of

30AuNP. The 13 nm gold nanoparticles (28.9 pmol) was dissolved in 100 mL of water.

To this solution, gold (III) chloride (22.34 μmol) was added followed by the addition of

trisodium citrate (13.5 μmol) and hydroquinone (23 μmol). This solution was stirred for 2

hours at room temperature.

Quantification of gold nanoparticles

The size of the gold nanoparticles was estimated from TEM/SEM images, samples of

which are shown below:

Figure S1 Samples of TEM/SEM micrographs used for measuring the size of AuNP. (a) 6AuNP (b) 13AuNP and (c) 30AuNP

UV-vis spectrometry was used to quantify all the AuNPs using the following

extinction coefficients (provided by Haiss et al. {Haiss:2007co}) at a wavelength of 450

nm

6AuNP: 1.26 x 107 M-1cm-1

13AuNP: 1.39 x 108 M-1cm-1

30AuNP: 1.96 x 109 M-1cm-1

mPEG functionalization of gold nanoparticles

The various AuNPs were coated with thiol functional mPEG (MW 800 g mol -1)

by incubating the nanoparticles with a specific amount of mPEG (6AuNP: 5000 eq.;

13AuNP: 20000 eq.; 30AuNP: 50000 eq.) under basic solution conditions (pH 9).

Purification of the 13AuNP and 40AuNP was accomplished by pelleting out the

nanoparticles using centrifugation (13AuNP: 13000 rpm for 30 min.; 40AuNP: 5000 rpm

for 30 min), while the 6AuNP required spin filtration units (MWCO 100 kDa) to remove

excess mPEG molecules.

Conjugation of oligonucleotides to gold nanoparticles

All sizes of gold nanoparticles were functionalized using the same salt-aging

protocol as described by Hurst et al.{Hurst:2006ea} The required amounts of gold

nanoparticles were incubated with DNA amounts as per the nanoparticle size (6AuNP: 75

eq., 13AuNP: 150 eq., 30AuNP: 1000 eq.). The ionic strength of the solution was

increased in 0.1 M increments to a final concentration of 1 M NaCl, with each increment

being added at 30-minute intervals. After salt aging, the nanoparticles were incubated

overnight, after which 10 eq. of thiol mPEG800 (relative to the amount of DNA) was

added to each sample and incubated for 1 hour. Excess DNA and mPEG800 molecules

were removed by pelleting out the nanoparticles by centrifugation (13 nm AuNP: 13000

rpm for 30 min, 30 nm AuNP: 5000 rpm for 30 min) and re-suspending them in borate

buffer (100 mM, pH 9.3). This process was repeated four times to ensure complete

removal of excess DNA. For the 6AuNP, excess DNA and mPEG800 was removed using

a spin filtration device (MWCO 100 kDa) as per the protocol of the manufacturer.

Quantification of the AuNP-DNA conjugates was accomplished using UV-vis

spectrometry.

Water-soluble quantum dots

Poly(ethylene glycol) methyl ether functionalized with dihydroxylipoic acid

(DHLA mPEG) was synthesized as per the protocol of Mei et al. {Mei:2009kt} For a

typical ligand exchange procedure, DHLA mPEG (20 μmol) was dissolved in 2 mL of

anhydrous ethanol, followed by the addition of oleic acid capped quantum dots (CdSexS1-

x/ZnS; core/shell) (1 nmol). The solution was purged with argon, and then incubated

overnight at 70 oC. The QDs were then precipitated out using a combination of hexanes

and chloroform, re-suspended in water and further purified using spin ultrafiltration

devices as per the protocol of the manufacturer. The QDs were quantified using UV-vis

spectroscopy, using the extinction coefficient of 2.1 x 105 M-1cm-1.

{Uddayasankar:2014kh}

Conjugation of oligonucleotides to QDs

Thiol functionalized oligonucleotide was modified with a peptide that consisted of

a hexahistidine moiety in the C-terminus and a maleimide group in the N-terminus. The

DNA was supplied with the thiol group protected as a disulfide, which required it to be

reduced using dithiothreitol before use. The required amount of DNA was incubated with

DTT (500 molar eq.) in PBS buffer. After 1 hour, excess DTT was removed by extracting

the aqueous solution with ethyl acetate (4 times). The reduced DNA was then incubated

with the peptide (10 eq.), which was dissolved in DMSO. After 24 hours, excess peptide

was removed using a NAP – 5 column, as per the manufacturer’s instructions. The DNA

in the resulting solution was quantified using UV-Vis spectroscopy, using the extinction

coefficient provided by the manufacturer.

The QDs were then incubated with the required amount of DNA-histag (usually

10 molar eq.) in borate buffer for at least 1 hour. Excess DNA was then removed using 4

rounds of spin ultrafiltration (MWCO 100 kDa).

Preparation of monofunctionalized nanoparticles

QDs

The DHLA-mPEG coated QDs were incubated with the DNA-histag (1 molar eq.) for

one hour in TB buffer. The required amount of magnetic beads was then added to the

solution (3.66 mg magnetic beads per nanomole of DNA) and vortexed for 1 minute to

capture the QD-DNA conjugates onto the magnetic beads. The magnetic beads were then

collected, washed twice with TB buffer and then incubated with a series of elution

solutions ([NaCl] (M): 0.15, 0.20, 0.225, 0.250 and 0.30). Verification of monoconjugate

elution was achieved using agarose gel electrophoresis (Figure S2a); with the fractions

containing the monoconjugates combined into one aliquot and quantified using UV-Vis

spectroscopy.

AuNPs

6AuNPs were first coated with bis(p-sulfonatophenyl)phenylphosphine to provide greater

colloidal stability. The AuNPs were then incubated with DNA (1 molar eq.) for one hour

in TB buffer. Thiol functional mPEG800 (1000 molar eq. to AuNP) was then added, and

the solution further incubated for 1 hour. The required amount of magnetic beads (3.66

mg magnetic beads per nanomole of DNA) was added to the solution and vortexed for 1

minute. The magnetic beads were then collected, washed twice with TB buffer and then

incubated with a series of elution solutions ([NaCl] (M): 0.10, 0.125, 0.150, 0.175, 0.20,

0.225 and 0.25). Verification of monoconjugate elution was achieved using agarose gel

electrophoresis (Figure S2b); with the fractions containing the monoconjugates combined

into one aliquot and quantified using UV-vis spectroscopy.

Figure S2 Preparation of monovalent nanoparticle – oligonucleotide conjugates. (a) Gel electrophoretic analysis of QDs functionalized with DNA, and monovalent conjugates purified using magnetic beads. Lane (i) depicts the initial mixture of QD-DNA valencies. Lanes (ii) – (vi) represent elution solutions of varying ionic strength ([NaCl]). (ii) 0.15 M (iii) 0.20 M (iv) 0.225 M (v) 0.250 M (vi) 0.30 M. (b) Gel electrophoretic analysis of AuNPs functionalized with DNA, and monovalent conjugates purified using magnetic beads. Lane (i) depicts the initial mixture of AuNP-DNA valencies. Lanes (ii) – (vi) represent elution solutions of varying ionic strength ([NaCl]). (ii) 0.10 M (iii) 0.125 M (iv) 0.150 M (v) 0.175 M (vi) 0.20 M (vii) 0.225 M (viii) 0.250 M

Optimizing the length of displacer DNA

Four different lengths of displacer DNA was immobilized onto 13AuNPs, and

subsequently incubated with 15 equivalents of QD525-probe DNA monoconjugates.

Fluorescence measurements were taken before and after the addition of target DNA

(>100 eq.), with the contrast ratio calculated as described in the experimental section of

the main manuscript.

Figure S3 Contrast ratio as a function of length of displacer DNA.

A lower contrast ratio is indicative of a higher background in the absence of target

DNA. This is a result of the low hybridization efficiency between the probe and displacer

strands that are immobilized on the nanoparticles. From Figure S3, displacer DNA

lengths less than 16 had lower hybridization efficiencies, as evidenced by the lower

contrast ratios. Displacer DNA lengths greater than 16 did not provide any further

improvements in hybridization efficiency.

Loading capacity for QDs around a central AuNP

A theoretical estimation of the number of QDs that may be accommodated on an

AuNP was obtained by assuming a closed packed arrangement of QDs (modeled as

spheres) around a single AuNP that was also modeled as a sphere. {Adams:1972eg} The

diameter of the QDs was approximately 5.0±0.5 nm. {Untitled:ul}

Table S1 Theoretical maximums for the number of QDs that may be closely packed around AuNPs of different diameters.

Size of gold nanoparticle, diameter (nm)

Maximum number of QDs

6.0±0.8 19±313±1 40±1031±4 190±70

Spectral overlap

Figure S4 UV-vis absorbance spectra (left axis) of three different sizes of gold

nanoparticles and photoluminescence spectra (right axis) of quantum dots with a peak

emission wavelength of 525 nm.

The spectral overlap integral was calculated using the following equation,

J ( λ )=∫0

⋈

ε A ( λ ) λ4 FD ( λ ) dλ

where J(λ) is the spectral overlap integral, εA is the extinction coefficient of the acceptor,

λ is the wavelength, FD is the area normalized fluorescence intensity.

Table S2 Calculated spectral overlap integral of QD525 emission with the absorption spectra of AuNPs of three different sizes.

Sample composition

Spectral overlap Integral, J (cm6 mol-1) (± 5 %)

QD525 – 6AuNP 1.20 x 10-7

QD525 – 13AuNP 1.60 x 10-6

QD525 – 30AuNP 2.80 x 10-5

Prediction of inner filter effect

The theoretical estimation of the inner filter effect was calculated using the

following relation ship.

Fraction of initial fluoresence observed=T A ×T E 1

where TA refers to the transmittance of radiation at the excitation wavelength, while TE

refers to the transmittance of radiation at the emission wavelengths.

Figure S5 Experimentally observed (red plots) and theoretically predicted (black plots) inner filter effect of AuNPs of three different diameters (a) 6 nm (b) 13 nm and (c) 30 nm.

Comparing proximal and distal configurations

Figure S6 Fluorescence intensity measurements of QD525 – AuNP conjugates for three different sizes of AuNPs in the absence (red bars) and presence (green bars) of target DNA. Results for two different configurations (distal and proximal) are presented.

Spectral overlap for QDs at different emission wavelengths

Figure S7 UV-vis absorbance spectra of 13AuNP and the fluorescence emission spectra of QD525, QD575 and QD630.

Table S3 Calculated spectral overlap integral of the emission of three different colors of QDs with the absorption spectra of a 13AuNP.

Sample composition

Spectral overlap Integral, J (cm6 mol-1) (± 5 %)

QD525 – 13AuNP 1.58 x 10-6

QD575 – 13AuNP 1.19 x 10-6

QD630 – 13AuNP 5.78 x 10-7

DNA functionalization of QDs

Figure S8 Confirmation of DNA immobilization on QDs. (a) Agarose gel electrophoretic image of (i) QDs and (ii) QDs functionalized with DNA. The samples were run on a 2 % agarose gel in 0.5xTBE as the running buffer at a field strength of 5.7 V cm -1. (b) UV-vis absorbance spectra of QDs and QDs functionalized with DNA.