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Probing Solid-State Nanopores with Light for the Detection of Unlabeled Analytes

Nanopore sensing has enabled label-free single-molecule measurements on a wide variety of analytes, including DNA, RNA, and protein complexes. Much progress has been made toward biotechnological applications; however, electrically probing the ion current introduces nonideal noise components. Here we further develop a method to couple an ionic current to a photon-by-photon counting of fluorescent signal from Ca2+-sensitive dyes and demonstrate label-free optical detection of biopolymer translocation through solid-state nanopores using TIRF and confocal microscopy. We show that by fine adjustment of the CaCl2 gradient, EGTA concentration, and voltage, the optical signals can be localized to the immediate vicinity of the pore. Consequently, the noise spectral density distribution in the optical signal exhibits a nearly flat distribution throughout the entire frequency range. With the use of high-speed photon counting devices in confocal microscopy and higher photon count rates using stronger light sources, we can improve the signal-to-noise ratio of signal acquisition, while the use of wide-field imaging in TIRF can allow for simultaneous quantitative imaging of large arrays of nanopores.


Rhod-2 -Ca 2+ binding constant measurements for different EGTA concentrations
Calcium chloride was titrated into a volume of 1M KCl to the final concentrations as indicated in Fig. S2. The concentration of Rhod-2 concentration was fixed at 5.6 µM, and fluorescence was measured at the confocal spot, positioned 100 µm above a glass coverslip. The experiment was repeated for three EGTA concentrations (10, 50, and 100 mM EGTA). The midpoint of each curve between background fluorescence and saturation was termed the kD for a given concentration of EGTA which competes for calcium binding with the calcium indicator Rhod-2. Higher concentrations of EGTA shift the midpoints toward higher concentrations of CaCl2.   and EGTA concentrations (cis).
A summary of the data shown in Figure S3 is given in Figure S4   Following previous studies 1, 2 we used the Poisson-Nernst-Planck equations to model ionic concentration distributions near nanopores. We numerically solve Poisson's equation, and the Nernst-Planck equation for each ionic species (potassium, calcium, and chloride), where V is the electric potential, F is the Faraday constant (~96,485 s·A/mol), ε is the dielectric constant permittivity of water (~7.09·10 -10 ·m -3 ·kg -1 ·s 4 ·A 2 ), and ci is the concentration, is the flux, Di is the diffusion constant, zCa is the charge number, and µCa is the electrophoretic mobility of each ion species i. The diffusion constant Di is calculated from the We solve for equilibrium spatially distributed steady-state free calcium in the presence of the chelating agent EGTA 4 according to where kD = 150 nM is the dissociation constant for EGTA-Ca 5 , [ ! ] is the total calcium concentration (numerically solved for with PNP), and [ ] is the concentration of EGTA. We solve for the concentration of calcium-bound Fluo-4 (again, following ref 4) according to where kD of Fluo-4 for Ca is 345 nM  is an experimentally determined concentration.
To model the total intensity acquired with TIRF illumination mode, we calculated the evanescent I(z) using: where n1 is the index of refraction of the trans chamber, n2 is the index of refraction of the cis chamber, and θ is the angle of incidence of the 488 nm excitation laser from cis to trans. We then integrate for total fluorescence (F): where L is 100 µm, the distance to the cis boundary in our numerical model.
The simulations were run with the potential difference across the pore ramped from -1V to 1V in 50 mV steps for each concentration of CaCl2. We then calculated F for each voltage and translocation Asymmetric distributions of calcium chloride, with CaCl2 in trans, were shown to both increase capture rate (Fig. S5) and slow down translocations (Fig. S6) without substantially changing the normalized blockade (Fig. S7) levels for 1 kbp DNA at 300 mV in a 4 nm pore relative to similar KCl gradients.

Comparison of Electrical versus Optical noise during long DNA translocations.
We compared the blocked level noise in the ion currents to those in the optical signal for a set of long DNA translocations (tD > 10 ms, N = 57). These events were acquired using a 8 kbp DNA, 2.8 nm pore at V = 1V). Electrical and optical events were acquired simultaneously, and for each event we measured the width at half maximum of the relative blocked ion current distributions during translocations. Our results (shown in Fig. S9) indicate that the optical signal as whole displayed more consistent average blocked levels, and a smaller dispersion overall than the electrical signal (the standard deviations in normalized blockade levels were 0.064 and 0.033, for the mean IB and FB of all events, respectively). This is consistent with the absence of flicker low frequencies noise in the spectrum of the optical signal, and may offer a significant improvement over electrical sensing when accurate determination of a current level is necessary.

Experimental Setup
A schematic of the optical and electrical components of the experiment is shown in Fig. S10.
Both confocal and TIRF modes can be used with two different laser lines, as explained in the Figure S11 | Electrical and optical measurements of Power Spectrum Distributions, measured simultaneously using 75 mM Ca 2+ , V =300 mV and 400 µM Rhod-2 (electrical signal filtered at 40 KHz using Butterworth filter).