A Modular Fluorescent Probe for Viscosity and Polarity Sensing in DNA Hybrid Mesostructures

Abstract There exists a critical need in biomedical molecular imaging and diagnostics for molecular sensors that report on slight changes to their local microenvironment with high spatial fidelity. Herein, a modular fluorescent probe, termed StyPy, is rationally designed which features i) an enormous and tunable Stokes shift based on twisted intramolecular charge transfer (TICT) processes with no overlap, a broad emission in the far‐red/near‐infrared (NIR) region of light and extraordinary quantum yields of fluorescence, ii) a modular applicability via facile para‐fluoro‐thiol reaction (PFTR), and iii) a polarity‐ and viscosity‐dependent emission. This renders StyPy as a particularly promising molecular sensor. Based on the thorough characterization on the molecular level, StyPy reports on the viscosity change in all‐DNA microspheres and indicates the hydrophilic and hydrophobic compartments of hybrid DNA‐based mesostructures consisting of latex beads embedded in DNA microspheres. Moreover, the enormous Stokes shift of StyPy enables one to detect multiple fluorophores, while using only a single laser line for excitation in DNA protocells. The authors anticipate that the presented results for multiplexing information are of direct importance for advanced imaging in complex soft matter and biological systems.


Transmission electron microscopy (TEM)
TEM images were acquired using a FEI Talos 120C at 120 kV operating voltage.

UV/Vis and fluorescence spectroscopy
UV/Vis and fluorescence measurements were carried out on an QE Pro from Ocean Optics equipped with the light source DH-2000-BAL and the temperature-controlled cuvette holder qpod 2e TM from Quantum Northwest for fiber optic spectroscopy, which enables to successively record UV/Vis spectra in 180° and fluorescence spectra in 90° ( Figure   S2). Time-resolved measurements were acquired using self-written MatLab scripts.

Plate reader
Fluorescence spectroscopic measurements with the DNA-microspheres were acquired using the Tecan Spark plate reader in top mode. The black 384 well plates from Costar Corning were used in which every well was filled with 30 μL of solution. The plate was kept at 25 °C during all the measurements.

Tunable NKT Photonics Laser
Samples were irradiated with an EXR-15 supercontinuum white light laser from NKT Photonics. The monochromatic irradiation wavelength was generated using the SuperK EXTEND-UV filter box from NKT Photonics.

pH measurements
pH measurements were carried out on a 907 Titrando from Metrohm.

Statistical analysis
Unless otherwise noted, all measurements were performed in triplicates (n = 3) to calculate the mean ± standard deviation. Linear regressions were calculated using OriginPro 2018G. Gaussian and mono-exponential fits were obtained by the Levenberg-Marquardt algorithm using the non-linear Origin Basic Functions (Gauss and ExpDec1) implemented in OriginPro 2018G. Fluorescence intensities were determined by the integration of the emission spectra using the tool curve_integ implemented in OriginPro 2018G. UV/Vis and fluorescence spectra were normalized to the absorption and the emission maximum, respectively.

Functionalization of StyPy
StyPy (20.2 mg, 46.2 µmol) was dispersed in DMF (4 mL) and mixed with DBU (5.9 µL, 14.1 mg, 92.6 µmol, 2.0 eq.) as well as with Boc-protected cysteamine. The yellow dispersion turned instantly to a clear orange solution, which was heated to 60 °C and stirred for 2 h. Subsequently, it was mixed with chloroform (20 mL) and washed with water (3 x 15 mL). The solvent was removed in vacuo yielding the desired product quantitatively.
The reduced ssDNA strand k (0.5 mM, 0.1 eq.) was mixed with StyPy (5 mM) in a mixture of DMSO : MilliQ water (80 : 20 vol-%) in presence of DBU (5 mM, 1 eq.). The resulting mixture was reacted at 37 °C for 1 d and DMSO was removed via freeze-drying. The resulting precipitate was extracted with MilliQ water and centrifuged to removed excess StyPy (4 x 21,000 g, 20 min). The resulting solution was freeze-dried and the product was obtained as a yellow solid.

Synthesis of p(A 20 -m-XL) and p(T 20 -n) DNA polymers
The synthesis of the multiblock DNA polymers were synthesized following our previous report. [1] The 5'-phosphorylated template and its corresponding ligation strand (see Table S2 Ultracentrifugal filters with a 10 kDa cut-off (Merck Millipore) and washed 3 times using TE buffer over the same filter.
The ssDNA concentrations were measured using a ScanDrop (Jena Analytic) spectrophotometer, and the solutions were diluted to 1 μM using TE buffer. The template synthesis was repeated multiple times, and a stock solution of the circular template was prepared to avoid batch-to-batch discrepancy.

Synthesis of the core-shell DNA microspheres
The core-shell particles were prepared by mixing the starting ssDNA multiblock polymers at the target concentration

Preparation of the single crystals
StyPy (1.31 mg) was dissolved in chloroform (1.78 mL) to obtain a concentration of c = 1.7 mM. The solution was filtered (0.2 µm pore size, PTFE) and transferred into glass tubes. The glass tubes were placed into a sealed vial, filled with iPrOH (10 mL). The solutions were left in the dark for 10 days, yielding the desired single crystals.  BPEA (9,10-bis(phenylethinyl)anthracene) with a quantum yield of Φ R = 1.00 in cyclohexane. [2] Φ Fl was calculated by [3] Φ l = Φ R X ( l) R ( l) 1 -10 R (425 nm) 1 -10 X (425 nm) where Int(Fl) is the integral of fluorescence, A(425 nm) the absorbance at 425 nm, and n the refractive index of the solvent, which was obtained from literature.
[4] The subscripts R and X denote the reference BPEA and StyPy, respectively. Both the UV/Vis and fluorescence measurements were repeated five times to obtain statistical confidence.