Peptide Sequences Were from Canpeptide Inc (Montreal, QC, Canada)

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):

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.

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,

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.

Prediction of inner filter effect

The theoretical estimation of the inner filter effect was calculated using the following relation ship.

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.

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.