Ultraefficient Cap-Exchange Protocol To Compact Biofunctional Quantum Dots for Sensitive Ratiometric Biosensing and Cell Imaging

An ultraefficient cap-exchange protocol (UCEP) that can convert hydrophobic quantum dots (QDs) into stable, biocompatible, and aggregation-free water-dispersed ones at a ligand:QD molar ratio (LQMR) as low as 500, some 20–200-fold less than most literature methods, has been developed. The UCEP works conveniently with air-stable lipoic acid (LA)-based ligands by exploiting tris(2-carboxylethyl phosphine)-based rapid in situ reduction. The resulting QDs are compact (hydrodynamic radius, Rh, < 4.5 nm) and bright (retaining > 90% of original fluorescence), resist nonspecific adsorption of proteins, and display good stability in biological buffers even with high salt content (e.g., 2 M NaCl). These advantageous properties make them well suited for cellular imaging and ratiometric biosensing applications. The QDs prepared by UCEP using dihydrolipoic acid (DHLA)-zwitterion ligand can be readily conjugated with octa-histidine (His8)-tagged antibody mimetic proteins (known as Affimers). These QDs allow rapid, ratiometric detection of the Affimer target protein down to 10 pM via a QD-sensitized Förster resonance energy transfer (FRET) readout signal. Moreover, compact biotinylated QDs can be readily prepared by UCEP in a facile, one-step process. The resulting QDs have been further employed for ratiometric detection of protein, exemplified by neutravidin, down to 5 pM, as well as for fluorescence imaging of target cancer cells.


B) Instrument and Methods
All moisture-sensitive reactions were performed under nitrogen atmosphere using oven-dried glassware. Dry solvents were obtained through an innovative technology solvent drying system. Evaporations were performed under reduced pressure on a rotary evaporator. The synthesis was monitored by TLC on silica gel 60 F254 plates on aluminum and stained by iodine. Flash column chromatography was performed on silica gel 60 A (Merck grade 9385). All 1 H and 13 C NMR spectra were recorded on Brucker DPX300 (500 MHz for 1 H, 125 MHz for 13 C) in CDCl 3 ). All chemical shifts were reported in parts per million (ppm) and the coupling constants were given in Hz.
High resolution mass spectra (HR-MS) were obtained on a Bruker Daltonics MicroTOF mass spectrometer. Infrared spectra were recorded on a PerkinElmer FT-IR spectrometer. UV-vis absorption spectra were recorded on a Varian Cary 50 bio UV-Visible Spectrophotometer over 200-800 nm using 1 mL quartz cuvette with an optical path of 1 cm or on a Nanodrop 2000 spectrophotometer (Thermo scientific) over the range of 200-800 nm using 1 drop of the solution with an optical path length of 1 mm. All centrifugations were carried out on a Thermo Scientific Heraeus Fresco 21 microcentrifuge using 1.5 mL microcentrifuge tubes at room temperature (unless stated otherwise). The QD purification was performed by Amicon ultra-centrifugal filter tubes with a cut-off MW of 30,000. Dynamic light scattering (DLS) was measured using Zetasizer Nano (Malvern) using the volume size distribution function. Atomic absorption spectra were recorded on a Perkin-Elmer Atomic Absorption Spectrometer AAnalyst 400, operating with an air-acetylene flame. Samples were prepared by dissolving the CdSe/ZnS QD prepared at 500 and 10000 LQMR in 1 M of HCl. Their Zn and Cd contents were obtained by using the corresponding absorbance and the standard calibration curves obtained with the ZnCl 2 or CdCl 2 solutions.
All fluorescence spectra were measured on a Spex Fluoro Max-3 Spectrofluorometer using a 0.70 mL quartz cuvette under a fixed excitation wavelength (λ EX ) of 450 nm. This wavelength corresponds to the absorption mimimum of the acceptor dye, minimizing the direct excitation of the dye acceptor. 1 An excitation and emission bandwidths of 5 nm and a scan rate of 120 nm/min over 480-800 nm range were used.
Confocal fluorescence imaging was recorded on Zeiss LSM-510 inverted laser scanning confocal microscope, (Germany) using at 488 nm excitation and collecting emission at above 570 nm. Flow cytometry was recorded on a BD LSRFortessa cytometer. The samples were excited at 488 nm and the emission was collected in the 570-585/42 nm band and the results were analysed using the FlowJo v10 software.

Step (i): Synthesis of MeO-PEG 750 -N 3 (2)
Mono methoxy-polyetheylene glycol with an average molecular weight of 750 (MeO-PEG750, 37.5 g, 50 mmol), THF (150 mL) and methanesulfonyl chloride (11.45 g, 100 mmol) were added in a 500 ml two-necked roundbottomed flask equipped with an addition funnel, septa and a magnetic stirring bar. Triethylamine (15 mL, 111 mmol) was added to the addition funnel. The reaction mixture was purged with nitrogen and cooled to 0 °C in an ice bath. Triethylamine was then added dropwisly to the reaction mixture through the addition funnel over a course of ~30 min. After that, the reaction mixture was warmed up gradually to room temperature (~20 °C) and stirred overnight. The reaction was monitored by silica gel TLC using CHCl 3 :MeOH =10:1 (vol/vol) as eluting solvent till the reaction was complete, R f(MsO-PEG750-OMe) = 0.65, R f (HO-PEG750-OMe) = 0.35. The reaction mixture was then diluted with H 2 O (50 ml) followed by addition of NaHCO 3 (3.125 g, 37 mmol). The resulting mixture was transferred to a separator funnel and extracted with CHCl 3 (60 mL × 3). The combined organic phase was evaporated by dryness on a rotary evaporator, giving the desired product as slightly yellowish oil, 40g (48.3 mmol, 96.6% yield).
Step (ii): Synthesis of OMe-PEG750-NH 2 (compound 3) N 3 -PEG 750 -OMe (8.0 g, ~10 mmol), EtOAc (150 mL) and 1 M HCl (25 ml, 25 mmol) were added to a 500 mL twonecked round-bottom flask. The reaction mixture was purged with N 2 and cooled to 0 °C by an ice-bath. Then triphenylphosphine (2.8 g, ~10 mmol) in 100 ml EtOAc was added dropwisely into the reaction mixture via an addition funnel under N 2 . The temperature was maintained to <5 °C during the addition. Once addition was complete, the reaction mixture was allowed to gradually warmed up to room temperature and stirred under N 2 overnight. The reaction mixture was transferred to a separation funnel and the biphasic solution was separated. The aqueous layer was collected and washed with EtOAc (100 mL×2) to remove any unreacted triphenylphosphine and formed triphenylphosphine oxide byproduct. The aqueous layer was transferred to a round-bottomed flask and cooled in an ice bath, into which KOH (13.5 g) was then added slowly. The mixture was stirred magnetically till all KOH was fully dissolved. The aqueous solution was then transferred to a separation funnel and extracted with CHCl 3 (60 mL × 5). The combined organic phase was dried over MgSO 4 (20 g, ~20 min) under stirring. After filting off MgSO 4 , the solvent was evaporated on a rotary evaporator, giving the desired compound as a light S-4 yellow oil in 59.2% yield (4.58 g). R f s (CHCl 3 :MeOH = 10:1, vol/vol) for R f (N3-PEG750-OMe) = 0.75, R f (NH2-PEG750-OMe) = 0.25. 1 H NMR (500 MHz, CDCl 3 ), δ (ppm), 2.87 (2H, -NH 2 ), 3.35 (s, 3H, -OCH 3 ), 3.6-3.9 (70H, m, CH 2 s in repeat PEG unit).
Thioctic acid (1.03 g, ~5.0 mmol) in 30 mL of CH 2 Cl 2 was then added dropwisely through the addition funnel over 30 min under N 2 . After the addition was complete, the reaction mixture was allowed to warm up to room temperature gradually and stirred overnight. A white precipitate was formed and filtered off through celite and the celite plug was rinsed with CHCl 3 . The combined solution was evaporated on a rotary evaporator. The residue was mixed with saturated NaHCO 3 and extracted with ether (100 mL × 2). The aqueous layer further extracted with CH 2 Cl 2 (100 mL × 2). The combined organic layers were dried over MgSO 4 . After filtration, the solvent was evaporated to yield the desired product as a yellow solid, weight 2. Step (i): Synthesis of LA-N, N-Dimethyl-1, 3-propanediamine.
Lipoic acid (3.0 g, ~15 mmol), triethylamine (1.47 g, ~15 mmol) and CH 2 Cl 2 (30 mL) were added to three-necked round bottom flask (250 mL) and stirred at 0°C for 30 min under a stream of N 2 . Methanesulfonyl chloride (1.67 g, ~15 mmol) was then added dropwisely through a syringe. The reaction mixture was allowed to slowly warm up to room temperature and left stirring for 5 hrs. After that, N,N-dimethyl-1,3-propanediamine (1.24, ~12 mmol) and triethylamine (0.61 g, ~6 mmol) in 20 mL CH 2 Cl 2 was slowly added to the reaction mixture and stirred at RT overnight under N 2 . The reaction mixture was transferred to a seperation funnel, washed with water (30 mL×2) and then saturated Na 2 CO 3 solution (100 ml). The organic layer was dried over Na 2 SO 4 and filtered. After evaporation of the solvent, the desired compound (1) was obtained as a yellow oil in 34.7% yield (1.48 g). 1  Step (ii): Synthesis of LA-ZW.
Compound 1 (1.48 g, ~5.2 mmol) was dissolve in 20 mL dry tetrahydrofuran (THF) and purged with N 2 for 30 mins, then 1,3-propanesultone (1.0 g, ~8 mmol) dissolved in 4 mL dry THF was slowly added and stirred for 3 days. A turbidity was formed instantly as the 1,3-propanesultone solution was added into the reaction mixture, an indication of forming LA-zwitterion which has low solubility in THF. Once the reaction was complete, the solvent was evaporated to give a crude product as a pale yellow solid. The crude product was washed with CHCl 3 (20 mL× 3) and further purified by HPLC to give the pure LA-ZW ligand in 23% yield. 1  Step (i) Synthesis of NHS-biotin.

Step (ii) Synthesis of LA-PEG600-biotin
LA-PEG600-NH 2 (0.29 g, ~0.37 mmol, containing ~13 PEG units synthesized previously), NHS-biotin (0.122 g, ~0.36 mmol) and DMF (8.0 mL) were placed in a 20 mL round-bottomed flask. The reaction vessel was purged with N 2 and cooled to 0 °C in an ice-bath under stirring. Then triethylamine (0.50 mmol) was added dropwisely through a syringe under N 2 . Once the addition was completed, the reaction mixture was allowed to gradually warm up to room temperature and stirred under N 2 overnight. The reaction mixture was filtered and the solvent was S-6 evaporated. The residue was purified by HPLC and the pure LA-PEG600-biotin fractions were collected. After evaporation of solvent, LA-PEG600-biotin was obtained as a waxy solid, weight 0.185 g (51.3% yield). 1

D1) Preparation of DHLA-zwitterion ligand capped QDs (QD-ZW)
A typical ligand exchange procedure for preparing QD-ZW is as follows: commercial hydrophobic CdSe/ZnS or CdSe/ZnSe/ZnS QD (1 nmol, 20 µL in hexane or tolune) was precipitated by adding 500 µL EtOH followed by centrifugation to remove any unbound free ligands. The QD pellet was then dispersed in 50 µL CHCl 3

D3) Protein preparation and prufication
The target protein, yeast SUMO (SUMO) protein and the anti-SUMO Affimers were expressed in BL21 (DE3) cells using isopropyl β-D-1-thiogalactopyranoside (IPTG) induction and purified by Ni-NTA resin (Qiagen) affinity chromatography according to the manufacturer's instructions. The detailed experimental procedures were described in our recent publication. 5

D4) Protein labeling 6
The anti-SUMO Affimer ( = 239,000 M -1 cm -1 ) and Affimer (ε 280nm =7904 M -1 cm -1 ) and the CF 280nm of 0.03 for the Alexa-647 dye, the average dye labeling ratio on per Affimer was calcualted as 1.08. The stock protein concentration was 86 µM. 6 Simiarily, the SUMO protein was labelled with Alexa-647 NHS ester using the same procedures under a dye: protein molar ratio of 6.4. After purification by using a G25 column as above, the average number of dyes labeled on each protein was determined as 0.80. The labeled protein stock concentration was 32.7 µM.
Neutravidin was also labelled with Alexa-647 NHS ester using the same procedures under a dye:protein molar ratio of 7. After purification by using a G25 column, the average Alexa-647 dye label per protein was determined as 1.67.

D5) Gel electrophresis analysis 6
2 or 5 µL of a DHLA-ZW capped CdSe/ZnS core/shell QD (QD-ZW, λ EM ~600 nm) prepared at a LQMR of 200, 500 or 1000 respectively was mixed with 18 or 15 µL of 60% glycerol in H 2 O. A QD-Affimer assembly was also prepared by mixing the QD-ZW prepared at a LQMR of 500 with 10 molar equivalent of the His 8 -tagged Affimer.
The resulting QD-Affimer conjugate was treated the same way as the above QD-ZW samples. Then 20 µL of each sample was loaded onto a 0.75% agrose gel in TAE buffer pH 8.3. Gel was run at 100 mV for ~30 min and QD was visualized under a UV illumination (λ = 365 nm).

D6) Cell culture and cell based studies 7
HeLa adherent epithelial cells derived from human cervical carcinoma were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 100 U mL -1 penicillin. The HeLa cells were trypsinized using trypsin-EDTA and maintained in a humidified incubator with 5% CO 2 at 37 o C.

Laser scanning confocal microscopy
S-8 2 mL of HeLa cells (1.5 × 10 5 cells mL -1 ) were cultured for 24 h followed by the treatment with 1 mL of the serum free DMEM with or without of the QD samples (50 nM). After incubation for 4 h, the cells were washed three times with Dulbecco's phosphate buffered saline. Hoechst 33342 was added to a final concentration of 5 µg mL -1 for nuclei staining. The cells were then imaged by laser scanning confocal microscopy (Zeiss LSM-510 inverted laser scanning confocal microscope, Germany). The QD was excited at 488 nm and the emission above 570 nm was collected.

D7) Atomic adsorption analysis of the QD Zn 2+ and Cd 2+ content
Two CdSe/ZnS QD samples after cap-exchange with the DHLA-zwitterion ligand at a LQMR of 500 and 10000 were prepared. They were then washed three times by pure water using a 30000 M.W. cut off filter tube to remove any unbound free ligand and etched Zn 2+ ions. The QDs were then dissolved in 1M HCl and then diluted with water before measurement. The Zn 2+ and Cd 2+ calibration curves were obtained by using the known concentration of the   than that prepared by photoligation (stable for 2 months), possibly due to the fact it was slightly aggregated (clustered) as revealed by our DLS and gel electrophoresis data (see Figure 2). Figure S6: Comparison of the normalised integrated fluorescence intensity for the CdSe/ZnS QD (λ EM ~600 nm) before (1, in CHCl 3 whose intensity was normalised to 100) and after cap-exchange with the DHLA-ZW ligand at a LQMR of 500 with (2, by the normal UCEP procedure with oxidized TCEP) and without (3) oxidized TCEP.  The original QD and the cap-exchanged QDs at low LQMRs (<500) appear to have bimodal size distributions (e.g. containing two species). In this case, the average D h was calculated by: Where D h 1 and D h 2 are the D h values, and A1% and A2% are the percentage areas (abundance %) of the two species obtained from the bimodal Guassian fit. The resulting data are summarised in Table S1.
Using the D h values obtained from the Guassian fits given in Table S1, the average particle hydrodynamic volume Therefore the V h (200) and V h (300) values are ~20 and ~8 times that of an isolated QD-ZW particle, respectively, suggesting that the QD forms small clusters or assemblies each containing a few to ~20 QDs after cap-exchange with the DHLA-ZW ligands under these low LQMRs of <500.
S-13    function of SUMO protein concentration, data were fitted to a linear function: Y = 0.0024 + 0.0014X, R 2 = 0.978. The limit of detection here was determined to be ~ 1 nM.

Figure S12.
Fluorescence spectra of the self-assembled QD-anti-SUMO Affimer sensor (final C QD = 10 nM) after incubation with the Alexa 647 labeled SUMO protein target (300 nM) in PBS (black), PBS containing 10% (red), 20% (blue) or 50% (pink) of human serum before (A) and after (B) correction of the human serum fluorescence background.
The above figure clearly shows that the QD-Affimer sensor prepared here is highly robust, it can specifically detect its target protein even under complex, clinically relevant conditions such as 50% human serum (HS).