Nanoscience in Fingerprinting – Caught Red-Handed
Team UCalgary BIOMOD 2018  Materials: Custom made oligonucleotides were purchased from Integrated DNA Technologies. Two lysozyme specific aptamer sequences were ordered: S1: 5’ AGC AGC ACA GAG GTC AGA TGG CAG CTA AGC AGG CGG CTC ACA AAA CCA TTC GCA TGC GGC CCT ATG CGT GCT ACC GTG AAT TTT TT G*G*G*G*G 3’ (lysozyme – aptamer dissociation constant Kd = 2.8 nM) [1] S2: 5’ G*G*G*G*G TTT TTT ATC AGG GCT AAA GAG TGC AGA GTT ACT TAG 3’ (lysozyme – aptamer dissociation constant Kd = 31 nM) [2]. For FRET experiments the following complementary sequences functionalized with Cy 5.5 fluorescent dye were ordered: For S1 aptamer-QD complex: 5’ /Cy5.5/ TTC ACG GTA GCA CGC ATA GGG 3’ For S2 aptamer-QD complex: 5’ CTA AGT AAC TCT GCA CTC TTT AGC CCT GAT /Cy5.5/ 3’. Tris was purchased from Bio-Rad. Sodium chloride (NaCl), hydrochloric acid (HCl), glycine, sodium phosphate dibasic (Na2HPO4), sodium tetraborate (Na2[B4O5(OH)4]), tellurium powder (Te), sodium borohydride (NaBH4), sodium hydroxide (NaOH), cadmium nitrate tetrahydrate (Cd(NO3)2 4H2O), 3-mercaptopropionic acid (MPA), cadmium chloride (CdCl2), and lysozyme from chicken egg white were purchased from SigmaAldrich and used without further purification. Buffer solutions: DNA storage buffer – 10 mM Tris-HCl, pH 8.0; DNA hybridization buffer – 10 mM Tris-HCl, 150 mM NaCl, pH 8.0. S1 binding buffer – 25 mM Tris, 192 mM glycine, 5 mM sodium phosphate dibasic, pH 8.3. S2 binding buffer – 15.7 mM sodium tetraborate, pH 8.4. Different composition of the binding buffers for two aptamers is based on the literature recommendations. Quantum dot synthesis [3]: CdTe Core Synthesis: NaHTe precursor was freshly prepared at room temperature by mixing 63.8 mg tellurium powder (0.5 mmol) with 75.6 mg sodium borohydride (2.0 mmol) in 10 mL deoxygenated dH2O at pH 9. The pH was adjusted by using 1 M NaOH solution prior to the reaction. The solution was continuously purged with nitrogen. The precursor solution was ready to use when the mixture turned transparent purple with no visible precipitate after 90 min. Cadmium nitrate solution was prepared by first adding 77.1 mg Cd(NO3)2 4H2O (0.25 mmol) and 37 µL MPA (0.425 mmol) to 50 mL of dH2O. The pH of the solution was adjusted to 12.0 using 1 M NaOH solution. The mixture was purged with nitrogen for 15 min, then 200 µL of NaHTe precursor was added at room temperature using a syringe. The reaction mixture was aged at 4°C overnight under inert atmosphere. The final ratio of Cd2+:MPA:Te was 1:1.7:0.04. CdS Shell Growth around CdTe cores and functionalization of quantum dots (QDs) with aptamers: CdTe/CdS core/shell quantum dots were prepared by adding 39.2 µL of 25 mM MPA solution and 17.5 µL of 25 mM CdCl2 solution to 700 µL of the prepared CdTe seeds in a 1.7 mL microcentrifuge tube. The mixture was vortexed and gently sonicated. Then, 350 µL of 100 nM aptamer solution was added. The mixture was gently vortexed. The pH of the solution was adjusted to 12 by addition of 1 M NaOH solution dropwise. The reaction solution was placed into an oven at 90 °C for different amounts of time. By varying the reaction time in the interval between 20 to 70 minutes, the emission maxima of the resulting aptamer functionalized CdTe/CdS core/shell QDs shifts from 550 to 630 nm. The aptamer functionalized core/shell nanoparticles were purified by first adjusting pH of the solution to 5.0 using 0.1 M HCl. Then, the particles were subjected to centrifugation at 14,000 rpm for 15 min followed by removal of the supernatant and resuspension of the pellet in dH2O. FRET experiments: Aptamer functionalized CdTe/CdS nanoparticles with an emission peak at 625 nm were selected for FRET experiments to confirm successful binding of the aptamers to the QD surface. The nanoparticles were first centrifuged at 14,000 rpm for 15 min, the supernatant was decanted, and the nanoparticles were resuspended in hybridization buffer. The complementary sequences functionalized with Cy5.5 fluorescent dye were added to the quantum dots at different concentrations. The final concentrations of the dye labelled oligonucleotides were 1, 3, 5, and 10 mM. Changes in the emission of QDs were monitored using a Varian Cary Eclipse spectrofluorimeter. The mixtures were excited at 400 nm and the emission was recorded between 500 and 750 nm to monitor changes in the emission of quantum dots. Print development: Two types of prints have been employed for development. Type 1: Lysozyme aqueous solution at a concentration of 10 mg/mL (714 µM) was used as ink to draw a pattern on a glass slide. The solution was air dried. Next, the slides with the drawn patterns were incubated with the solution containing either aptamer functionalized or bare (no aptamer attached) quantum dots with respective binding buffers. Type 2: A latent fingerprint was deposited on an epoxy functionalized glass slide. Prior to pressing the fingertip against the surface of the slide, the finger was rubbed against the forehead. Next, the slides with the deposited fingerprints were incubated with the solution containing either aptamer functionalized or bare (no aptamer attached) quantum dots with respective binding buffers. All the images were taken under continuous irradiation of the samples with UV light. References: [1] Tran, D. T.; Janssen, K. P. F.; Pollet, J.; Lammertyn, E.; Anné, J.; Van Schepdael, A.; Lammertyn, J.; Tran, D. T.; Janssen, K. P. F.; Pollet, J. Selection and Characterization of DNA Aptamers for Egg White Lysozyme. Molecules 2010, 15 (3), 1127–1140 [2] Automated Selection of Anti-Protein Aptamers by J. Colin Cox and Andrew D. Ellington