DNA Nanostructure Assembly
1. Add 12 𝜇L of each oligo (stock: 100 𝜇M) for P1 (Star oligo 1 – 12, Extension oligo A – E, F, and G) and P2 (Star oligo 1 – 12, Extension oligo A – E, and F’) in separate tubes, 72 𝜇L of 5x TAEMg buffer and deionized water to reach a total reaction volume of 360 𝜇L with a final concentration of 3.3 𝜇M for each oligo in 1x TAEMg1 (40 mM Tris, 20 mM acetic acid, 2 mM EDTA, and 12.5 mM magnesium acetate, pH 8).
2. Vortex the samples, divide and transfer each sample into 4 PCR tubes, and place the samples in a thermal cycler such as the MiniAmp Plus Thermal Cycler (Applied Biosystems, A37834).
3. Incubate samples at 95 oC for 5 minutes then cool to 90 oC. Ramp down from 90 oC to 60 oC at 0.4 oC/min, 60 oC to 26 oC at 0.03 oC /min, then snap cooled to 4 oC indefinitely.
4. Prepare 50 mL 0.5x TBE in an Erlenmeyer flask, weigh out and add 1 g of agarose to the 0.5x TBE buffer to make 2 % gel, and microwave for 15-30 seconds at a time for approximately 2 minutes and 30 seconds until the agarose is completely dissolved. Let the agarose cool, add 5 𝜇𝐿 of 10,000x GelRed to agarose solution, and gently mix to avoid creating bubbles. Pour the agarose solution into the gel tray, insert comb, and let agarose solidify for 20 minutes. Note: the volume of agarose and gel stain needed depends on the size of the gel plate used. For this protocol a gel tray with the following dimensions (10 cm x 7 cm) was used.
5. Pool the DNA nanostructure probes from the four PCR tubes after the assembly, mix 0.5 𝜇L of each DNA nanostructure probe with 9.5 𝜇L of deionized water and 2 𝜇L of 6x Loading buffer (ThermoFisher Scientific, R0611) for gel characterization. Load the samples on 2 % agarose gel, run the gel at 70 V for approximately 1 hour, and visualize on Gel imaging workstation (Azure biosystems, C150). The remaining probe can be stored at 4 oC.
DNA Nanostructure Purification
1. Take 90 𝜇L of assembled probe (P1/P2), add 14 𝜇L of 5x TAEMg, 15 𝜇L of 10x GelRed, 25 𝜇L of 6x Loading Buffer (ThermoFisher Scientific, R0611), and 26 𝜇L of deionized water. Vortex the samples and load into wells of 5% Mini-PROTEAN TBE polyacrylamide pre-cast gels (BioRad, 4565013). Run the gel in 0.5x TBE buffer at 100 V on ice for 3 hours.
2. Take the gels out of cassettes and visualize using gel imager (Azure biosystems, C150) UV plate.
3. Identify the band of interest and excise with a razor blade.
4. Place the excised gel slices (containing products of interest) in custom extraction apparatus or D-Tube Dialyzer Maxi (MWCO 6-8 kDa, Millipore, 71509-3) tubes, and apply 100 V in 1x TBE buffer to electrophoretically extract the DNA samples out of the gel slices.
5. Take the supernatant (0.5 mL to 3 mL) from the extraction tubes, and perform buffer exchange into 1x TAEMg using 100 kDa Amicon Ultra-0.5 Centrifugal Filter Units (Millipore Sigma, UFC510096). This should concentrate the sample into ~ (35 𝜇L).
Quantification of Nanoprobes
6. Quantify the purified probes by taking 2 𝜇L of the sample and performing nucleic acid concentration measurements using a UV-Visible spectrophotometer for small sample volumes such as the Take3 micro-volume plate accessory of the EPOC 2 spectrophotometer (BioTek, BTEPOCH2).
7. Take equimolar concentrations of purified DNA nanostructure probes, where the probe concentrations are fixed at either 15 nM or 20 nM, and add various amounts of junction strands, ranging from 0.2 nM (0.01:1 junction:probe) to 400 nM (20:1 junction:probe) for dumbbell assembly.
8. Incubate for up to two days (48 h) at room temperature.
9. Confirm the quality of dumbbell formation on 2% agarose gel as shown in Figure 1.
Nanopore Characterization Measurements
10. Take 4 𝜇L of the assembled dumbbell samples, add 35.5 𝜇L of 3.6 M LiCl and 0.5 𝜇L 2kbp dsDNA fragments (ThermoFisher Scientific, SM1701).
11. Vortex the sample and load it into the nanopore flow cell (cis side)2,3.
12. Apply -100 mV and sense on a nanopore for 10 - 30 minutes, anticipated results are shown in Figure 2.
Paramagnetic Bead Ab Conjugation
1. Add 234 𝜇g of primary antibody (Fitzgerald Industries International, 10C-CR2151M4) to 100 kDa Amicon Ultra-0.5 Centrifugal Filter Unit (Millipore Sigma, UFC510096).
2. Add Bead Conjugation Buffer (Quanterix, 101357) to the filter unit to reach a final volume of 500 𝜇L.
3. Centrifuge at 14,000 g for 5 minutes, discard flow-through, and wash the samples by adding 450 𝜇L Bead Conjugation Buffer.
4. Repeat the 450 𝜇L washes three times.
5. Invert the filter unit membrane and centrifuge into a new tube at 1,000 g for 2 minutes.
6. Rinse the filter unit membrane by adding another 50 𝜇L of Bead Conjugation Buffer, centrifuge at 1,000 g for 2 minutes into the same tube.
7. Collect the sample and measure the protein concentration using the Take3 micro-volume plate accessory of the EPOC 2 spectrophotometer (BioTek, BTEPOCH2).
8. Add enough Bead Conjugation Buffer to dilute the sample to 0.3 mg/mL.
9. Store the antibody sample on ice.
10. Take 300 𝜇L (8.5 x 108) of carboxylated paramagnetic beads sized 2.7 𝜇m (Quanterix, 103612) and resuspend by vortexing.
11. Briefly centrifuge the sample using the pulse spin setting of a small 8 sample rotor bench top centrifuge and place solution on magnet for > 1 minute, remove supernatant.
12. Add 600 𝜇L of the Bead Wash Buffer (Quanterix, 101355), and vortex to resuspend beads.
13. Repeat 600 𝜇L washes three times using Bead Wash Buffer ensuring to vortex the beads each time, then remove the buffer after the samples have been placed on the magnet for >1 min.
14. Add 600 𝜇L of Bead Conjugation Buffer (Quanterix, 101357), vortex to resuspend beads, then use the magnet to retain the beads and remove the buffer.
15. Repeat 600 𝜇L washes three times using Bead Conjugation Buffer (Quanterix, 101357) ensuring to vortex the beads each time, then remove the buffer after the samples have been placed on the magnet for >1 min.
16. Add 570 𝜇L of the Bead Conjugation Buffer and store on ice.
17. Add 100 𝜇L of the Bead Conjugation Buffer to 1 mg vial of EDC (ThermoFisher Scientific, A35391), close lid and vortex.
18. Add 30 𝜇L of the EDAC to the washed paramagnetic beads and incubate on shaker (VWR, PTR-35) for 30 minutes to allow for bead activation.
19. Pulse spin using tabletop centrifuge (VWR, 76269-064), and wash activated paramagnetic beads with 600 𝜇L Bead Conjugation Buffer (Quanterix, 101357), then place on magnet for >1 min and remove supernatant.
20. Add prepared buffer exchanged primary antibody to activated paramagnetic beads.
21. Incubate for 2 hours on shaker.
22. Pulse spin, place paramagnetic bead/primary antibody solution on magnet, collect supernatant and label as “supernatant”.
23. Perform one wash with 600 𝜇L Bead Wash Buffer (Quanterix, 101355), collect supernatant and label as “wash 1”.
24. Measure protein concentrations of supernatant and wash 1 using EPOC 2 spectrophotometer (BioTek, BTEPOCH2). Compare values to total amount of antibody loaded to estimate total antibody coated and percentage antibody coated.
25. Perform another wash with Bead Wash Buffer (Quanterix, 101355), then discard the supernatant.
26. Add 600 𝜇L of Bead Blocking Buffer (Quanterix, 101356) and incubate for 30 minutes on Multi-Functional Tube Rotator (VWR, PTR-35) at 5 rpm to keep particles in suspension.
27. Perform one wash using 600 𝜇L of Bead Wash Buffer (Quanterix, 101355).
28. Perform two more washes using 600 𝜇L of Bead Diluent (Quanterix, 100458), resuspend in 600 𝜇L Bead Diluent and store at 4 oC.
Secondary Antibody Streptavidin Conjugation
1. Take 62.5 𝜇L (100 𝜇g) of secondary antibody (Maine Biotechnology Services, MAB130P) and add 37.5 𝜇L of 1x PBS to have secondary antibody at 1 𝜇g/𝜇L in 100 𝜇L.
2. Add 10 𝜇L of Modifier reagent (Abcam, ab102921) to 100 𝜇L of secondary antibody.
3. Add the above 100 𝜇L sample to lyophilized Streptavidin Mix (Abcam, ab102921), pipette up and down to have material fully dissolved.
4. Incubate at room temperature for 3 hours.
5. Add 10 𝜇L of Quencher (Abcam, ab102921) to the reaction.
6. Incubate at room temperature for 30 minutes.
7. Confirm the quality of the conjugation using native gel, Mini-PROTEAN TGX gels (Bio-rad, 456-1093) in 1x Tris/Glycine Buffer at 100 V
8. Store at 4 oC.
Assay Procedure
1. Take 72 𝜇L (7.2×107) of prepared bead-capture antibody conjugates, add Sample Diluent (Quanterix, 101359) to a total volume of 500 𝜇L.
2. Place the tube on the magnet, and remove supernatant.
3. Perform two more 500 𝜇L washes.
4. Add various amounts (0.8 fM to 1.2 nM) of recombinant TSH (rTSH) (BiosPacific, J11030) and bring the volume up to 500 𝜇L using Sample Diluent or diluted human serum sample.
5. Incubate for 1 hour at room temperature on Multi-Functional Tube Rotator (VWR, PTR-35) at 5 rpm.
6. Place the tube on the magnet for >1 min, and remove the supernatant.
7. Add 500 𝜇L of Wash Buffer 1 (Quanterix, 100486), and incubate for 5 minutes on Multi-Functional Tube Rotator (VWR, PTR-35) at 5 rpm.
8. Place the tube on the magnet for >1 min, and remove the supernatant.
9. Add 500 𝜇L of Wash Buffer 1 (Quanterix, 100486), and incubate for 10 minutes on Multi-Functional Tube Rotator (VWR, PTR-35) at 5 rpm.
10. Place the tube on the magnet for >1 min, and remove the supernatant.
11. Add 500 𝜇L of Wash Buffer 1 (Quanterix, 100486), and incubate for 15 minutes on Multi-Functional Tube Rotator (VWR, PTR-35) at 5 rpm.
12. Place the tube on the magnet for >1 min, and remove the supernatant.
13. Resuspend in 500 𝜇L of Sample Diluent (Quanterix, 101359) containing 6 nM secondary antibody-streptavidin conjugate. OPTION 1: for the amplification assay, add 10 𝜇𝐿 of AuNP amplification complex to a final volume of 50 𝜇L in Sample Diluent (Quanterix, 101359).
14. Incubate for 1 hour on Multi-Functional Tube Rotator (VWR, PTR-35) at 5 rpm at room temperature.
15. Perform three 30 sec washes with 1x Wash Buffer 1 (Quanterix, 100486), and resuspend in 500 𝜇L of Sample Diluent (Quanterix, 101359). OPTION 1: for theamplification assay, skip this step.
16. Add 12 nM of biotinylated ssDNA junction strand. OPTION 1: for the amplification assay, skip this step.
17. Incubate for 15 min at room temperature on Multi-Functional Tube Rotator (VWR, PTR-35) at 5 rpm. OPTION 1: for the amplification assay, skip this step.
18. Perform three 30 sec washes with 1x Wash Buffer 1 (Quanterix, 100486), and resuspend in 500 𝜇L of 1x TAEMg buffer containing 0.025 % Tween-20. OPTION 1: for the amplification assay, resuspend in 20 𝜇L of 1x PBS containing0.025 % Tween-20.
19. Apply UV using a 3W LED flashlight (LIGHTFE, UV301D) at a distance of 1 cm for 20 minutes to release the ssDNA junction strand. OPTION 1: for the amplification assay, release the junction strand by incubation for 45 minutes in 0.2 M DTT (ThermoFisher Scientific, A39255) and 0.3 M NaH2PO4 buffer in a final volume of 30 𝜇L.
20. Place the samples on the magnet for >1 min and transfer the supernatant to 3 kDa Amicon Ultra-0.5 Centrifugal Filter Unit (Millipore Sigma, UFC500396).
21. Perform buffer exchange and concentrate the ssDNA junction strands into 35 𝜇L of 1x TAEMg containing 0.025 % Tween-20.
22. Add the purified shooting star probes at either 15 or 20 nM final concentrations.
23. Incubate at room temperate for up to 2 days for dumbbell assembly.
OPTION 1: Gold Nanoparticle Amplification Scheme
DTT Reduction
1. Take thiolated ssDNA oligos and perform three washes of 1x PBS using 3 kDa Amicon Ultra-0.5 Centrifugal Filter Unit (Millipore Sigma, UFC500396) to have final concentration of 10 𝜇M in 1x PBS.
2. Prepare 0.3 M NaH2PO4 containing0.2 M DTT solution (ThermoFisher Scientific, A39255).
3. Mix 250 𝜇L of the buffer exchanged DNA oligo and 250 𝜇L of 0.3 M NaH2PO4 and 0.2 M DTT.
4. Incubate at room temperature for 2 hours (unless otherwise stated incubations were done at room temperature on the benchtop.).
5. Take a NAP-5 column (GE Healthcare, 17185601), remove all caps and let storage buffer flow through.
6. Equilibrate the NAP-5 column with 10 mL deionized water.
7. Add the reduced DNA sample to the column and allow sample to fully enter membrane.
8. Place column on top of a new tube.
9. Add 1 mL deionized water to elute purified sample.
10. Reduce the volume to 200 𝜇L using 3 kDa Amicon Ultra-0.5 Centrifugal Filter Unit (Millipore Sigma, UFC500396), confirm and adjust the final concentration of the sample to to 25 𝜇M with deionized water.
11. Split the sample into 50 𝜇L aliquots and store at -20 oC.
Preparation of Amplification complex. (AuNP Functionalization with antibody and ssDNA oligos)
1. Take 960 𝜇L of 30 nm AuNP (Cytodiagnostics, G-30-20), add 40 𝜇L 0.1 M borate buffer to adjust the solution to pH 9.
2. Add 4 𝜇g of secondary antibody (Maine Biotechnology Services, MAB130P) to a 1.7 mL tube, and add the adjusted AuNP solution to the tube with the secondary antibody4,5.
3. Incubate at room temperature for 30 minutes.
4. Add 25 𝜇L of reduced ssDNA oligos and incubate for 1 hour.
5. Salt age to a final concentration of 0.15 M NaCl by adding 6 additions of 1 M NaCl every 30 min.
6. Incubate at 4 oC overnight (optional pause point).
7. Add 50 𝜇L of 10 % BSA and incubate for 5 minutes.
8. Centrifuge at 4,500 g for 15 minutes. Discard the supernatant.
9. Add 950 𝜇L of 1x PBS containing0.025 % v/v Tween-20, resuspend by gentle shaking or vortexing.
10. Repeat steps 8 – 9 two times.
11. Add 150 𝜇L of 1x PBS containing0.025 % v/v Tween-20.
12. Store at 4 oC.