Quantifying Nanomolar Protein Concentrations Using Designed DNA Carriers and Solid-State Nanopores

Designed “DNA carriers” have been proposed as a new method for nanopore based specific protein detection. In this system, target protein molecules bind to a long DNA strand at a defined position creating a second level transient current drop against the background DNA translocation. Here, we demonstrate the ability of this system to quantify protein concentrations in the nanomolar range. After incubation with target protein at different concentrations, the fraction of DNA translocations showing a secondary current spike allows for the quantification of the corresponding protein concentration. For our proof-of-principle experiments we use two standard binding systems, biotin–streptavidin and digoxigenin–antidigoxigenin, that allow for measurements of the concentration down to the low nanomolar range. The results demonstrate the potential for a novel quantitative and specific protein detection scheme using the DNA carrier method.

. Raw statistics of DNA carrier translocation events after incubation with target proteins S1. Nanopore pulling programs and size distribution Detailed nanopore fabrication methods are described previously 1 . Two slightly different programs are used for biotin-streptavidin system and the digoxygenin-antidigoxigenin system with the pulling programs listed in Table S1. With lower heat, program 2 produces nanocapillaries of larger size as determined by the distribution of ionic current drop caused by double stranded DNA translocation shown in Figure S1. It is clear that the dsDNA current drops from program 2 are smaller indicating the nanopore size is larger.  Table S1. Parameters of capillary pulling programs. Figure S1. Distributions of dsDNA translocation ionic current drop from nanopores produce by pulling program 1 (a) and 2 (b).

S2. Occupied translocation event threshold determination.
To differentiate the blank and occupied DNA carrier translocation event, a threshold factor (I/I DNA ) based on the dsDNA current amplitude was set to determine the protein binding peak, where I is the peak current drop amplitude in the searching window and I DNA is the S3 current drop amplitude caused by dsDNA translocation. The fraction of occupied events decreases with the increasing threshold factor as shown in Figure S2.
For the blank carrier events (black), the fraction drops from the factor of 1 and reaches 0 at the factor of about 1.5. Since this control measurement should ideally show no occupied fraction, all events detected between the factor 1 and 1.5 are background noise or caused by DNA with complex folds or knots. As for the fully occupied events (red and green), the occupied fraction, which ideally should be around 1, starts to drop for factors of about 1.4 and larger, meaning some of the occupied events start to be lost. The optimal threshold factor varies among different nanopores: with smaller diameter nanopores and lower noise (green), higher threshold factor can be set without losing occupied detection while in the in red, smaller factor is needed. We choose the factor of 1.4 as a compromise to keep the occupied events for most of the nanopores and filter out most of background noise.

S3. Electrophoresis gel shift assay
A 38 bp duplex with one oligonucleotide modified with 5' three thymine and biotin or digoxigenin was titrated against the respective binding protein. The DNA duplexes were incubated with the target proteins at different concentration for 30 minutes at room temperature before gel loading. The incubations were carried out in 100 mM NaCl, 2 mM MgCl 2 buffered with 10 mM Tris-HCl in the same condition as for the nanopore measurements. 3% agarose gel and 1×TAE as running buffer was used at the voltage of 100 V for 1 H. The gel is then stained with Gel Red (Biotium) in all the assays. As a reference to the nanopore measurements, EMSA is used to measure the differences among the three samples. The concentration ratio (c Binding site / c Oligo ) is used here given the divalent nature of the antibody. Unlike the short DNA duplex which could bind to both sites of one antibody molecule, we did not observe a significant number of DNA carrier dimers at lower protein concentrations. This shows that for the 7.2 kbp DNA carrier, it is not possible for two carriers to be connected by one antibody. The weak band seen above the dominant oligo-antibody complexes band is probably an antibody with only one oligo attached.

S4. Fluorescent polarization measurement
A 16mer (16 thymine) DNA oligo modified with 6FAM (5') and digoxigenin (3') was used. 8nM DNA modified oligo was titrated against anti-dig of the concentrations from 1 nM to 120 nM. The incubations were carried out in 100 mM NaCl, 2 mM MgCl 2 , 10 mM Tris-HCl with and without 4 M LiCl. Setup runs of the FP assay were performed in 96-well plates and read on a CLARIOstar plate reader (BMG Labtech) with a 488/520 FP filter. The binding curve in high salt (4 M LiCl) buffer shows that the binding curve is not significantly affected by this high salt and that the binding fraction is still saturated at a few nM. This can be expected given that shape complementarity (and not electrostatic interaction) was explained as the primary reason for the anti-digoxigenin-digoxigenin affinity based on an analysis of crystal structures. 2 Figure S4.
where P 0 and L 0 are the initial concentration of protein and ligand added. The binding fraction can be calculated as: All the fittings in this work were done with the model described here using OriginPro9.

S5. Minimum sampling size discussion
The translocation event recording is considered as a Binomial distribution. The confidence interval E is obtained where p is the occupied fraction, z * is the z value of the chosen confident level and N is the sample size. The minimum N can be estimated with N ൌ z * ଶ pሺ1 െ pሻ MOE ଶ where MOE is the margin of error or the accuracy we aim to maintain. 4 When 1.96 is used as the z value for 95% confident level, the equation produces the minimum N for 10% and 1% accuracy shown in Figure S5. The event numbers needed for different occupied fraction to achieve certain margin of error are listed in table S2.

S6. Translocation event rate comparison
The translocation rates of the proteins with and without DNA carrier under the same experimental conditions were compared. It should be noted that for protein only translocations that it is likely that some translocations are missed due to bandwidth restrictions. As shown in Figure S4(a), the normalized event rate of both streptavidin and anti-dig translocation alone increases as a function of the bias voltage applied which is consistent with the previous literature 5 .
The event detection threshold was set as 50 pA for streptavidin and 70 pA for Anti-Dig. The event rate of unfolded DNA carrier is slightly higher: 0.08 ± 0.004 Hz /nM (Biotin carrier) and 0.10 ± 0.013 Hz /nM (Digoxigenin carrier) at 600 mV voltage compared to the protein translocation event rate of 0.052 ± 0.008 Hz /nM (streptavidin) and 0.057 ± 0.007 Hz /nM (anti-dig) under the same voltage of 600mV. In addition, the event rates of both biotin carrier and digoxigenin carrier are independent from the incubated protein concentration ( Figure  S4b).

S7. Raw statistics of DNA carrier translocation events after incubation with target proteins
Detailed statistics of translocation event numbers and nanopore information is listed in Table  S2 (biotin-streptavidin system) and