Rhodium-Complex-Functionalized and Polydopamine-Coated CdSe@CdS Nanorods for Photocatalytic NAD+ Reduction

We report on a photocatalytic system consisting of CdSe@CdS nanorods coated with a polydopamine (PDA) shell functionalized with molecular rhodium catalysts. The PDA shell was implemented to enhance the photostability of the photosensitizer, to act as a charge-transfer mediator between the nanorods and the catalyst, and to offer multiple options for stable covalent functionalization. This allows for spatial proximity and efficient shuttling of charges between the sensitizer and the reaction center. The activity of the photocatalytic system was demonstrated by light-driven reduction of nicotinamide adenine dinucleotide (NAD+) to its reduced form NADH. This work shows that PDA-coated nanostructures present an attractive platform for covalent attachment of reduction and oxidation reaction centers for photocatalytic applications.


Synthesis of CdSe seeds
The CdSe seed synthesis was adapted from a known procedure in literature. 2 3.00 g trioctylphosphine oxide (TOPO), 0.28 g octadecylphosphonic acid (ODPA) and 0.06 g CdO were placed in a 25-mL-threeneck-flask. The reaction was performed under an inert atmosphere and constant stirring. The flask was heated to 80 °C to melt the chemicals and evacuated to remove water in the chemicals. Once no more gas emerged from the solution, the flask was heated to 120 °C and kept evacuated for 30 min. After that, the flask was purged with N2. The flask was then heated to 320 °C, upon which the solution turned colorless due to the complexation of Cd-ODPA, and then cooled down to 120 °C. Then, a vacuum was applied to remove water, a side product of the complexation of Cd-ODPA; after gas formation in the reaction mixture stopped (at least 2 h), then, the flask was purged with N2 and heated to 340 °C. Next, 0.058 g Se dissolved in 0.36 g trioctyl phosphine (TOP) were injected. After the injection, the heating was immediately stopped, and the flask was cooled down by N2 airflow to accelerate the cooling speed. After the temperature of the mixture was cooled to 90 °C, 5 mL of toluene was injected into the mixture. The seeds were cleaned five times by centrifugation with 10 mL toluene in 10 mL methanol and then dissolved in toluene. The diameter of the CdSe seeds was determined from the energetic position of the absorption peak with the lowest energy 3 to be 5 nm.

Synthesis of the CdSe@CdS nanorods (NR)
The CdSe@CdS nanorods were synthesized according to a reported protocol in literature. 4 3.35 g trioctylphosphine oxide (TOPO), 1.08 g octadecylphosphonic acid (ODPA), 0.207 g CdO, and 0.06 g npropylphosphonic acid (PPA) were placed in a 25-mL-three-neck-flask. The reaction was performed under an inert atmosphere and constant stirring. The flask was heated up to 80 °C until the reaction mixture melted and evacuated to remove residual water from the mixture. Once gas formation stopped, the flask was heated to 120 °C and kept evacuated for 30 min. After that, the flask was flooded with N2. The flask was then heated to 320 °C, upon which the solution turns colorless due to the complexation of Cd-ODPA, and then cooled down to 120 °C. Next, a vacuum was applied until gas formation in the reaction mixture stopped (at least 2 h) to remove water, which is a side product of Cd-ODPA complexation. Then, the flask was flooded with N2 again and heated up to 340 °C. Next, 1.5 g TOP and 0.05 g sulfur dissolved in 0.60 g TOP were injected. After 20 s, 2 mg of CdSe seeds (diameter = 2.2 nm) dissolved in 0.5 g TOP were injected. The reaction was allowed to stir for 10 min until the color of the solution turned from red to orange. 5 mL toluene was injected into the mixture once the temperature dropped below the flashing point of toluene to stop solidification of the mixture. After precipitation with 10 mL methanol, rods were cleaned five times by centrifugation with 6 mL n-hexane, 2 mL nonanoic acid, and 2 mL octylamine in 10 mL methanol. Next, the size of the rods was excluded by centrifugation at 4200 rpm for 30 min with 10 mL toluene and 8 mL isopropanol to obtain rods with lengths of circa 50 nm. This was repeated two times in total. The NR were then dispersed in toluene for further investigation.
Ligand exchange with 11-mercaptoundecanoic acid (MUA) The ligand exchange with MUA was performed according to a protocol in literature. 5 250 mg MUA were dissolved in 25 mL methanol and tetramethylammonium hydroxide pentahydrate was added until the solution reached pH 11 (circa 200 mg). 20 mg NR (dried from its toluene solution under vacuum) were added into this mixture and stirred for 2 h. When the NR were fully dispersed, approximately 35 mL of toluene was added as non-solvent until NR precipitated. The mixture was then centrifuged at 6000 rpm for 20 min, the supernatant discarded, and the precipitate was redispersed in degassed water.

Synthesis of [(ipphCOOH)Rh(Cp*)Cl]Cl
In a 50 mL round bottom flask, 19.0 mg (0.0307 mmol, 1 eq.) [Rh(Cp*)Cl2]2 and 20.9 mg (0.0613 mmol, 2 equiv.) ipphCOOH were suspended in a 15 mL 1:1 (v:v) MeOH:DCM mixture. After ultrasonicating for 10 min, the mixture was stirred for 16 h at room temperature. After almost complete evaporation of the solvent, the desired compound was precipitated from the solution using Et2O. After filtration, 37.8 mg (0.0582 mmol, 95%) of the compound were obtained as yellow solid.    Polydopamine coating of the nanorods 50 µL of the nanorods solution (3 mg/mL) were diluted with 400 µL of Tris-HCl buffer (0.1 M, pH = 8.5) in an 1.5 mL Eppendorf tube and 50 µL of dopamine solution (1 mg/mL in MilliQ water) were added. The Eppendorf tube was wrapped in aluminum foil to prevent light exposure. Then, the reaction solution was vortexed for 24 h at 1000 rpm at room temperature. For purification, the reaction solution was filtered through a centrifuge filter at 4000 g to remove small molecule residuals like free dopamine. The coated nanorods remained in the filter and were redispersed in MilliQ water. The filtration was repeated twice. As centrifuge filters Millipore centrifugal filters units (Amicon Ultra -0.5 mL, Ultracel -100k) with a cut-off of 100 kDa and a centrifuge (Heraeus Fresco 2, Thermo Fisher Scientific) was used. For further functionalization the remaining PDA-coated nanorods were redispersed in phosphate buffer (0.1 M, pH = 8.5) while they were redispersed in MilliQ for storing.

Functionalization of coated nanorods with catalyst and PEG
For functionalization 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 1.2 eq.), Nhydroxysuccinimide (NHS, 2 eq.), methoxypolyethylene glycol 5,000 acetic acid (PEG, 0.03 eq.) and 0.1 mL of [(ipphCOOH)Rh(Cp*)Cl]Cl dissolved in methanol (3 mg/mL) were added to 0.5 mL of PDAcoated nanorods in phosphate buffer (0.1 M, pH = 7) in an Eppendorf tube. The Eppendorf tube was wrapped in aluminum foil to prevent light exposure and the solution was vortexed for 24 h at 1000 rpm at room temperature. For purification, the reaction solution was filtered three times through a centrifuge filter at 4000 g to remove small molecules while the functionalized nanorods remained in the filter and were redispersed in MilliQ water. The same centrifuge filters as for the coated nanorods were used.
Steady-state absorption spectroscopy: Absorption spectra of nanorods (both bare and coated) were recorded in a quartz cell (d = 1 cm) using a JASCO V780 UV-Visible/NIR spectrophotometer (JASCO GmbH, Pfungstadt, Germany). All measurements were performed in a wavelength range of 300 nm to 700 nm. A cuvette with pure solvent was always measured as a reference.
Steady-state photoluminescence spectroscopy: Photoluminescence spectra of nanorods (both bare and coated) were recorded in a quartz cell (d = 1 cm) using an FLS980 photoluminescence spectrometer (Edinburgh Instruments Ltd., Livingston, UK) in a 90° geometry. An excitation wavelength of 450 nm was used to record photoluminescence spectra covering a wavelength range of 500 nm to 650 nm. The optical density of the dispersions was set to 0.05 to avoid inner filter effects and reabsorption of photoluminescence. Absolute photoluminescence quantum yields of bare nanorods were recorded at an excitation wavelength of 450 nm using a barium sulfate coated integrating sphere. 6,7 The integrating sphere was mounted on the fluorimeter with the entry and output ports of the sphere located in 90° geometry from each other in the plane of the spectrometer. Photoluminescence was recorded from 425 to 700 nm. The colloidal samples were held in a five-face transparent quartz cuvette located in the center of the integrating sphere and the optical density at the excitation wavelength was adjusted to 0.1. As a reference sample, a cuvette filled with pure water was recorded under identical conditions. The quantum yield of coated nanorods were estimated by measuring both bare and coated nanorods under similar conditions, i.e., same excitation parameters and a similar absorbance at the excitation wavelength, without using the integrating sphere.
Thermal NAD + reduction: In order to test for the catalytic activity of immobilized Rh-catalyst, a 10 µg/mL concentrated suspension of either cNR-Rh-PEG or cNR-PEG in 50 mM aqueous NaHCO2 was prepared in an argon filled glovebox and transferred into a sealable cuvette filled with 2.5 mL of the reaction solution. The solution, which additionally contained 250 µM NAD + , was heated to 45 °C using a water bath. After certain time intervals the reaction progress was analyzed using emission spectroscopy on a JASCO FP-8500 fluorescence spectrometer (exc = 340 nm).
Irradiation setup for photocatalysis: For the NAD + reduction, 250 µM NAD + and 10 µg/mL of the PDAcoated nanorods functionalized with the Rh catalyst and PEG were dissolved in the respective solvent. As solvents either demineralized water, a water methanol mixture (1:1), water containing 0.12 M trimethylamine (TEA) and 0.1 M NaH2PO4 or Tris-HCl buffer (25 mM, pH = 7.5) were used. All reactions were performed with 2.5 mL of the reaction solution at room temperature inside gas-tight quartz glass cuvettes (d = 10.0 mm, Hellma) under argon atmosphere. The reactions were irradiated for 180 minutes. The irradiation setup consisted of a custom-made reactor and one blue light emitting LEDstick (max = 466 nm, 45-50 mW/cm 2 ) in the center of the reactor on which the cuvettes were placed. 8 The photocatalysis runs were analyzed via UV-vis spectroscopy on a JASCO V-670 UV-vis-NIR spectrophotometer or emission spectroscopy on a JASCO FP-8500 fluorescence spectrometer.
Quantification of produced NADH: For determination of the amount of NADH produced during photocatalysis, a calibration curve using commercially available NADH (Sigma Aldrich, 97% purity) was S8 recorded as follows: Samples of different NADH concentrations (0 µM, 2.5 µM, 5.0 µM, 10.0 µM, 15.0 µM, 20.0 µM, 25.0 µM and 30.0 µM) were prepared in 2.5 mL deionized water under ambient conditions. Emission spectra were recorded using λexc = 340 nm. The intensity of the emission maximum at 462 nm was plotted against the molar concentration of NADH to obtain the required calibration curve. (see Figure S18). The known masses of NADH were divided by the mass of the photocatalytic system (10 µg/mL) and plotted against the measured intensities (see Figure S21). The data was fitted with a square function giving = 2 • 10 −8 2 + 0.0004 with being the intensity and the mass NADH per mass of photocatalytic system. The produced mass of NADH per mass of the photocatalytic system was calculated using the given equation and the measured intensities. Energy-dispersive X-ray spectroscopy (EDX): The EDX spectrum was recorded on an Hitachi SU8000 scanning electron microscope (Hitachi High-Technologies Europe GmbH, Krefeld) with a Bruker Quantax EDX device. For the measurements a primary energy of 8 keV, a tilt angle of 0°, a take off angle of 30° and as detector a Bruker XFlash 5010 (fifth generation, 10 mm 2 detector area) was used. The data was analyzed using the Bruker Quantax Esprit 2.3 software.
Transmission electron microscopy (TEM): TEM images of nanorods after synthesis and before ligand exchange were recorded in scanning mode (STEM) using a JEM-ARM200F NEOARM (Jeol) operating at 80 kV, equipped with spherical aberration corrector, bright field (BF), annular bright field (ABF) and annular dark field (ADF) detectors. Several images were recorded and 640 particles were analyzed regarding their length and width using FIJI (ImageJ v. 1.53c). 9 The TEM images for determination of the coating thickness and after the treatment with HAuCl4 were recorded with a Tecnai F20 (Field Electron and Ion Company, FEI) using an accelerating voltage of 200 kV.
X-ray photoelectron spectroscopy (XPS): XPS was performed in an ultra-high-vacuum (<2 × 10 -10 mbar) multiprobe system (Scienta Omicron) using a monochromatized X-ray source (Al Kα, 1486.7 eV) and an electron analyzer (Argus CU) with a spectral resolution of 0.6 eV. The spectra were calibrated using the Au 4f7/2 peak (84.0 eV) and the Si 2p peak (SiO2, 103.6 eV), respectively and fitted using Voigt functions (30:70) after background subtraction. Zeta-potential: 60 µL of cNR, cNR-PEG or cNR-Rh-PEG (0.2 mg/mL) were diluted to 1 mL in an aqueous solution of 1 mM KCl. The dilution ratio was adjusted, if the count rate was not sufficient. The zeta potential was derived from the electrophoretic mobility of the particles and measured using a Zeta Nanosizer ZS (Malvern Instrument) with 1 mL disposable folded capillary cells (Zeatasizer Nano series, Malvern). Each measurement was performed in triplicates.  The XRD pattern ( Figure S8) of NR shows (102) and (103) reflections at 36.6° and 47.9°, respectively, characteristic for the wurtzite structure. Additionally, the intense (002) reflection is characteristic for CdSe@CdS NR obtained from the seeded-growth method due to the preferred growth along this facet. 4 The XRD pattern of cNR is qualitatively the same, but some reflections, in particular the (102) and (103) reflections, are less intense, while the (100) reflection appears more pronounced.   The observed features in the high-resolution N 1s (E) and O 1s (F) spectra of the cNR match well to the literature of polydopamine films. 10 After the Rh-catalyst and the PEG are attached to the cNR, the spectra change slightly. The nitrogen atoms from imidazole, amide and pyridine in the Rh catalyst contribute to the species at lower binding energy, whereas both nitrogen atoms from the pyridine groups coordinated with the Rh atoms can be ascribed to the signal at higher binding energies (see Figure S13 C). In the O 1s spectrum (D), the component ascribed to O-C and SiO2 increased due to two different reasons. On the one hand, the addition of PEG leads to a higher amount of O-C bonds and on the other hand the oxygen signal from the SiO2 substrate is stronger in comparison to (F), as confirmed by the more intense Si 2p signal (not shown).