Chirality enriched carbon nanotubes with tunable wrapping via corona phase exchange purification (CPEP)†

Single-walled carbon nanotubes (SWCNTs) have unique photophysical properties and serve as building blocks for biosensors, functional materials and devices. For many applications it is crucial to use chiralitypure SWCNTs, which requires sophisticated processes. Purification procedures such as wrapping by certain polymers, phase separation, density gradient centrifugation or gel chromatography have been developed and yield distinct SWCNT species wrapped by a specific polymer or surfactant. However, many applications require a different organic functionalization (corona) around the SWCNTs instead of the one used for the purification process. Here, we present a novel efficient and straightforward process to gain chirality pure SWCNTs with tunable functionalization. Our approach uses polyfluorene (PFO) polymers to enrich certain chiralities but the polymer is removed again and finally exchanged to any desired organic phase. We demonstrate this concept by dispersing SWCNTs in poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co(6,6’-{2,2’-bipyridine})] (PFO-BPy), which is known to preferentially solubilize (6,5)-SWCNTs. Then PFO-BPy is removed and recycled, while letting the SWCNTs adsorb/agglomerate on sodium chloride (NaCl) crystals, which act as a toluene-stable but water-soluble filler material. In the last step these purified SWCNTs are redispersed in different polymers, surfactants and ssDNA. This corona phase exchange purification (CPEP) approach was also extended to other PFO variants to enrich and functionalize (7,5)-SWCNTs. CPEP purified and functionalized SWCNTs display monodisperse nIR spectra, which are important for fundamental studies and applications that rely on spectral changes. We show this advantage for SWCNT-based nIR fluorescent sensors for the neurotransmitter dopamine and red-shifted sp defect peaks ðE* 11Þ. In summary, CPEP makes use of PFO polymers for chirality enrichment but provides access to chirality enriched SWCNTs functionalized in any desired polymer, surfactant or biopolymer.


Introduction
One of the great challenges in nanoscience is to obtain materials of well-defined structure and size. 1,2 Polydisperse nanomaterials can lead to wrong conclusions in fundamental studies on structure-function relationships but also affect performance in applications. Therefore, progress in synthesis and purification of nanomaterials is crucial to advance the whole nanotechnology field. An important class of nanomaterials are single walled carbon nanotubes (SWCNTs). 3,4 Semiconducting SWCNTs are fluorescent in the near infrared (nIR) region and the emission wavelength depends on their structure described by the chirality or chiral index (n,m). [5][6][7][8][9] The nIR fluorescence of SWCNTs has been used for example for imaging, biosensing or single-photon generation. [10][11][12][13][14] Even though the synthesis of specific SWCNT chiralities has seen progress over the past years typical SWCNT starting materials still contain multiple chiralities. [15][16][17] Therefore, purification and separation of SWCNTs remains a major area of research.
Here, we report a general but straightforward route to chirality enriched SWCNTs with tunable wrapping. We make use of the high-selectivity of PFO polymers for certain chiralities, remove/recycle the PFO polymer and obtain a chirality enriched SWCNT material stabilized in a NaCl filler/scaffold to avoid strong aggregation. This material is then used for different non-covalent functionalization approaches and the advantages of monodisperse SWCNTs are shown for different applications.

Results and discussion
To get access to nearly chirality-pure SWCNTs with any desired non-covalent functionalization we developed a three-step approach.
It makes use of the high selectivity of polymers of the PFO family for certain SWCNT chiralities. Then the PFO-polymer is removed and recycled, while the purified SWCNTs can be further surface modified via standard tip sonication. This corona phase exchange purification (CPEP) approach makes the organic phase independent of the surfactant and polymer used for purification, which is crucial for most applications of SWCNTs.
The CPEP approach (see Fig. 1) is based on the high selectivity of PFO-BPy for (6,5)-SWCNTs. A CoMoCAT SWCNT sample is dispersed with PFO-BPy in toluene via shear force mixing and separated from larger bundles by centrifugation. 36 The highly enriched PFO-BPy-(6,5)-SWCNTs are precipitated with isopropyl alcohol (4 : 1, toluene : isopropyl alcohol), while the excess PFO-polymer stays in solution after centrifugation (Fig. 2a). Redispersion of the SWCNT pellet in toluene and repeated precipitation decreased the concentration of the free PFO-BPy to a minimum, which can be monitored by UV-Vis-nIR absorbance spectroscopy (Fig. 2a). The purified nanotubes were then transferred to a solvent-resistant filter loaded with NaCl crystals, which act as toluene-resistant, but later on water-soluble filler/scaffold (see ESI Fig. S1 †). Washing with hot toluene removed the remaining PFO-BPy from the (6,5)- Fig. 1 Schematic of the corona phase exchange purification (CPEP) process. The process is based on selective dispersion of (6,5)-SWCNTs by PFO-BPy or certain SWCNT chiralities by other members of the PFO family in toluene. PFO-BPy is removed by adding isopropyl alcohol, which precipitates the SWCNTs, while the majority of PFO-BPy stays in solution and can be recycled. The (6,5)-SWCNT pellet is transferred to a solvent-resistant filter equipped with NaCl crystals, which act as water-soluble filler material during removal of remaining PFO-BPy residues. The (6,5)-SWCNT flakes are then available for any functionalization approach e.g. with ssDNA, SDBS or biopolymers. Fig. 2b), while SWCNTs adsorbed/agglomerated on and in between the filler. After drying and removing the NaCl, the purified (6,5)-SWCNT flakes were ready to be resuspended with any desired polymer. Addition of trifluoroacetic acid (TFA) to support PFO removal from SWCNTs decreased the nIR-fluorescence of redispersed (6,5)-SWCNTs and was therefore not used (see ESI Fig. S2 †).

SWCNTs (
Combining the PFO-BPy supernatants furthermore enabled us to recycle and reuse the (expensive) PFO-BPy polymer for nanotube dispersion (see Fig. 2c).
The SDBS sample shows a strong blue shift of the S 11 and S 22 transition, which could indicate a more effective debundling in the CPEP processed sample. Absorbance spectra were fitted ( Fig. 3d-f ) to quantify the ratio of SWCNT-chiralities. The S 11 transitions showed that PFO-BPy dispersion yielded ∼94% pure (6,5)-SWCNTs while resuspending in e.g. ssDNA did not change this enrichment significantly (87% for (AT) 15 -(6,5) and 90% for (GT) 10 -(6,5)-SWCNTs after CPEP), which is mainly attributed to differences in background and not the chirality composition itself. The yield of SWCNT dispersion could be further improved, by using a higher-power shear force mixer 36 instead of the customary homogenizer for shear force dispersion that was used here for CPEP.
We also evaluated the nanotube lengths after the CPEP of the two exemplaric ssDNA-SWCNTs and compared it with the one from the PFO-BPy-(6,5)-SWCNTs (see ESI Fig. S3 †). The mean length from the PFO-BPy SWCNTs was ∼1460 nm (SE 723 nm), which is slightly smaller than the length reported in literure. 36 Both ssDNA-(6,5)-SWCNT were in mean nearly half as long with 770 nm (SE 358 nm) for (AT) 15 and 757 nm (SE 347 nm) for (GT) 10 . This results shows, that the resuspension of the purified (6,5)-SWCNTs obviously decreases the lateral mean size, while the absolute lengths is larger than the one, known from dispersing unpurified SWCNTs with ssDNA. 41 The success of the purification procedure can be further visualized in SWCNT fluorescence spectra. Fig. 4a shows a 2Dfluorescence spectrum of ssDNA modified SWCNTs without further purification. It shows the emission of (6,5)-SWCNTs and other chiralities such as (6,4), (7,5) or (8,3). In contrast, the PFO-BPy modified SWCNTs (Fig. 4b) contain mainly (6,5)-SWCNTs as expected. The exchange to ssDNA and aqueous solution does not significantly ( Fig. 4c and d) change the spectra. This result highlights how effective CPEP is to remove other chiralities and obtain monodisperse samples with a desired functionalization.
For biosensing application, it is of special interest whether CPEP processed and redispersed (6,5)-SWCNTs are still functional in terms of nIR-fluorescence responses to analytes. Fig. 5a shows a fluorescence spectrum of CPEP (GT) 10 -(6,5)-SWCNTs and its increase after addition of dopamine [100 nM]. This type of functionalization is known to make SWCNTs responsive to the important neurotransmitter dopamine. 41,45 A single fluorescence peak of such sensors is essential for multiplexing approaches that use multiple SWCNT chiralities or other fluorophores. Therefore, monochiral samples could improve multiplexed sensing and imaging approaches for example to exploit differences in responsiveness by different chiralities. 50,62 Another application that requires non-congested spectra is defect engineering. 13,63,64 These approaches lead new emission features and even to single-photon emitting SWCNTs and fun- damental insights into the mechanism rely on well-defined and unambiguous spectra. CPEP processed SWCNTs can be used to introduce defects as shown in Fig. 5b. The introduction of (PhNO 2 ) aryl sp 3 -defects into the SWCNT surface causes a second, red-shifted fluorescence peak at ∼1165 nm. Related studies with PhNO 2 defected SDBS-(6,5)-SWCNTs report E * 11 fluorescence maxima at ∼1145 nm. 65 Especially the tunable corona modification with different ssDNA sequences could help to improve SWCNT-based chemical sensing and imaging. Importantly, it expands the possibilities of non-covalent functionalization schemes to purified SWCNTs closer to covalent functionalization schemes. 66,67 In addition to the modification of highly enriched (6,5)-SWCNTs with different ssDNA sequences and surfactants (see ESI Fig. S4 †), it is possible to exchange the corona to biocompatible PEG-polymers (ESI Fig. S5 †). Hereby, the purified SDBS-and PEG-(6,5)-SWCNTs showed a minor nIR-fluorescence peak at ∼1120 nm, which cannot be monitored in the complimentary spectra from the raw nanotube material. Either other chiralities superimpose these features (see also ESI Fig. S6 †), or they get enhanced due to the purification process.
Besides the advantages of well-defined fluorescence features CPEP processed samples appear to be brighter compared to similar non-purified samples with the same functionalization (ESI Fig. S11 †). (GT) 10 -(6,5)-SWCNTs display around 70% higher fluorescence intensities at the same concentration/absorption, which could be either explained by their increased length or the absence of quenching impurities.
The high monodispersity of CPEP processed SWCNTs is based on the specific dispersion of certain SWCNT chiralities by polyfluorene polymers. 37,38 This step relies also on the raw nanotube material and its ratio of semiconducting to metallic nanotubes and the containing chiralities. 70 Therefore, by choosing the appropriate SWCNT material and PFO-polymer for dispersion, CPEP can grant access to a brought range of enriched SWCNTs with desired surface modifications. This approach can be especially relevant for fundamental studies and applications of SWCNTs that require sophisticated surface chemistry beyond standard surfactants. Consequently, applications such as biosensors, imaging, drug delivery and defect engineering can profit from CPEP.

Conclusions
Chirality pure SWCNTs are necessary both for fundamental studies and biomedical applications. In this work we present a novel route to chirality enriched SWCNTs with tunable functionalization. It uses the selective dispersion of SWCNTs in PFO-polymers but removes/recycles them again and exchanges them to any non-covalent modification. These chirality enriched SWCNTs are functional and can be employed in applications for which the functionalization plays a crucial role and monodisperse spectra are essential such as singleemission fluorescence sensors and defect peak engineering.

Purification protocol
Highly enriched (6,5)-SWCNTs were obtained based on the dispersion protocol from Graf et al. 36 50 mg PFO-BPy (American Dye Source) were dissolved in 100 mL toluene by gently warming the solution. 25 mg chirality enriched (6,5)-SWCNTs (Sigma Aldrich, Product No. 773735) were added and placed for 20 h shear-force mixing (Homogenizer PT3100, Polytron) in a water bath, in order to keep the temperature during the surface modification process constantly at 20°C. The following centrifugation step (15 min/20 500g/15°C) yielded the highly enriched PFO-BPy-(6,5)-SWCNT stock solution. Two times 8 mL of the PFO-BPy-(6,5)-SWCNT solution were mixed with 2 ml isopropyl alcohol and centrifuged 15 + 5 min (20 500g/ 15°C). This ratio of toluene to isopropyl alcohol led to a loss of the colloidal stability of the nanotubes, while the majority of the PFO-PBy stayed in solution. Lower ratios of isopropyl alcohol to toluene resulted in an incomplete precipitation of the SWCNTs, while a much higher ratio increased the amount of precipitated polymer. The (6,5)-SWCNT pellet was resuspended in 8 ml toluene and bath sonicated for 2 min. Afterwards, two more rounds of precipitation with isopropyl alcohol, centrifugation and resuspension were performed. The progress of PFO-BPy removal was followed by its UV-Vis absorption. The pellet was further transferred to a solvent resistant filter (2.2 NY, Ciro), which was equipped with 200 mg powdered NaCl crystals, which act as toluene-resistant but water-soluble filler/scaffold material. In the following washing step, the nanotube pellet was washed with 5 × 600 µl (90°C) hot toluene, which removed the remaining PFO-BPy from the sample. The SWCNT loaded NaCl crystals were dried under vacuum and dialyzed for 24 h in a 1 kDa dialysis bag (Spectra/ Por®) against ddH 2 O. The resulting (6,5)-SWCNT flakes were transferred to a reaction tube, ready for further surface modification with a desired water-soluble surfactant. For ssDNA modification, 150 µl PBS and 125 µl (2 mg ml −1 ) oligonucleotide solution were tip sonicated (30%, 45 min, Fisher ScientificTM Model 120 Sonic Dismembrator), followed by 10 min centrifugation at 10 000g. The same sonication conditions were used for SWCNT dispersion in 275 µl 0.2% SDBS aqueous solution. The residual PFO-BPy solution was evapor-ated and reused for SWCNT modification, while the undispersed SWCNT material was also recycled. 36 SWCNT dispersion with PFO was performed similarly using 1 mg ml −1 PFO in toluene.

UV-Vis-nIR absorption and nIR-fluorescence spectroscopy
UV-Vis-nIR absorbance spectra of the SWCNT conjugates were acquired with a JASCO V-670 device in a 10 mm path quartz cuvette. Fitting of absorption spectra was based on the approach by Pfohl et al. 71 In short, a background profile of the form e −bλ was fitted to the absorption spectra and subtracted. The normalized spectra were then fitted in Python to a function consisting in the sum of nine Lorentzians using a standard least squares fit, with the Trust Region Reflective algorithm.
1D and 2D nIR-fluorescence spectra were acquired with a Shamrock 193i spectrometer (Andor Technology Ltd, Belfast, Northern Ireland) connected to an IX53 microscope (Olympus, Tokyo, Japan). Excitation was performed with a Monochromator MSH150, equipped with a LSE341 light source (LOT-Quantum Design GmbH, Darmstadt, Germany). 200 µl of aqueous SWCNT conjugates were placed in a 96-well plate, while toluene-based samples were analyzed in a glass vial.

Conflicts of interest
There are no conflicts to declare.