Robust and specific ratiometric biosensing using a copper-free clicked quantum dot–DNA aptamer sensor

A robust and copper-free clicked quantum dot–DNA sensor for sensitive and ratiometric detection of specific DNA and protein targets at the pM level is reported.


S3
Polyetheylene glycol with an average molecular weight of 600 (PEG600) (30 g, 50 mmol), THF (50 ml) and methanesulfonyl chloride (13.2 g, 115 mmol) were added in a 250 ml two-necked round-bottomed flask equipped with an addition funnel, septa and a magnetic stirring bar. Triethylamine (17.2 ml, 123 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 (addition takes~30 min). After that, the reaction mixture was warmed up gradually to room temperature (~20°C) and stirred overnight. The product was checked by TCL on silica gel with CHCl 3 :MeOH =10:1 (vol/vol) as elution solvent, R f (MsO-PEG 600 -OMs) = 0.62, R f (HO-PEG 600 -OH) = 0.36. The mixture was then diluted with H 2 O (66 ml) and NaHCO 3 (4.13 g, 0.049 mol) was added. The resulting mixture was transferred to a separator funnel and extracted with CHCl 3 (60 ml × 3).
The combined organic phase was evaporated by driness on a rotary evaporator, yileding slightly yellowish oil, 40g, with~90% yields.
The product (40 g), sodium azide (10 g, 0.154 mol), THF (50 ml), H 2 O (50 ml) and NaHCO 3 (0.5 g) were then added to two-necked round-bottomed flask equipped with a distilling head with a round-bottomed flask as a solvent trap. The solvent trap was cooled with an ice-bath. The biphasic reaction mixture was heated under N 2 to distill off the THF. The reaction mixture was then refluxed for~8 hrs (or overnight). After the reaction mixture was cooled to room temperature, it was transferred to a separator funnel. The product was extracted with CHCl 3 (100 ml × 5).
The product was checked with TCL using CHCl 3 : MeOH =10:1 (vol/vol) as elution solvent, R f (N 3 -PEG 600 -N 3 ) = 0.75. The combined organic layers were dried over Mg 2 SO 4 (~20 g, for~30 min) with stirring. The Mg 2 SO 4 was filtered off and the dried organic layer was evaporated to dryness on a rotary evaporator. A pale brown oil (33.21g, yield: 97%) was obtained . 1  Step 2: Amine transformation of one terminal azide group, synthesis of N 3 -PEG 600 -NH 2 (compound 3) N 3 -PEG 600 -N 3 (8.012g, 11.7 mmol), EtOAc (60 ml) and 1 M HCl (27 ml, 27 mmol) was added in a 250 ml twonecked round-bottomed flask equipped with an addition funnel, septa and a magnetic stirring bar. Triphenylphosphine (3.36 g, 12.8 mmol) dissolved in 45 ml EtOAc was transferred to the addition funnel. The reaction vessel was purged with N 2 and cooled to 0°C in an ice-bath while stirring. The triphenylphosphine solution was then added dropwisely under N 2 . The temperature was maintained to below 5°C during the addition. Once the addition was completed, the reaction mixture was gradually warmed up to room temperature and stirred for~8 h (or overnight) under N 2 . The reaction mixture was then transferred to a separatory funnel and the biphasic solution was separated. The aqueous layer was collected and washed with EtOAc (60 ml × 2). The aqueous layer was transferred to a round-bottomed flask with a magnetic stirring bar. The solution was cooled in an ice bath. KOH (13.5 g) was then added slowly to the aqueous solution and the mixture was stirred until the KOH is dissolved. The aqueous solution was transferred into a separatory funnel and extracted repeatedly with EtOAc (60 ml × 5). The combined organic layer was dried over MgSO 4 (20 g for~20 min) with stirring. After filting off MgSO 4 , the solvent was evaporated to dryness on a rotary evaporator, yielding a light yellow oil, 6.5 g, yield:~80%. R f s (CHCl 3 :MeOH = 5:1, vol/vol) for N 3 -PEG 600 -NH 2 = 0.09, N 3 -PEG 600 -N 3 = 0.8. 1 H NMR (500 MHz, CDCl3): d 3.6-3.9 (m), 3.52 (t, 2H, J= Hz), 3.40 (t, 2H, J= 4.8 Hz), 2.87 (t, 2H, J =5.0 Hz) N 3 -PEG 600 -NH 2 (6.5 g, 9.8 mmol), 4-(N,N-dimethylamino)pyridine (0.2407 g, 1.977 mmol), N,N'-dicyclohexylcarbodiimide (2.07 g, 10.07 mmol) and CH 2 Cl 2 (60 ml) were added into a 250 ml round-bottomed flask equipped with a magnetic stirring bar and an addition funnel. The mixture was kept at 0°C in an ice bath. Thioctic acid (2.02 g, 9.8 mmol) dissolved in 32 ml of CH 2 Cl 2 was transferred to the addition funnel. Thioctic acid solution was added dropwisely over 30 min under N 2 while stirring. After the addition was complete, the reaction mixture was allowed to warmed up to room temperature gradually, and the mixture was stirred overnight. The mixture was then filtered off through celite and the celite plug was rinsed with CHCl 3 . The solvent was evaporated on a rotary evaporator.
100 ml H 2 O was added to the residue and the mixture was washed with diethyl ether (100 ml × 2). The aqueous layer was then saturated with NaHCO 3 . The product was extracted with CHCl 3 (100 ml × 3). The combined organic layers were dried over MgSO 4 (for 20 min). After filtering off the MgSO 4 , the solvent was evaporated on a rotary evaporator. The residue was then purified on a silica gel chromatograph (5.0 cm i.d. × 20 cm length) using 20:1 (vol/vol) CHCl3: MeOH as the eluent solvent. Each fraction was checked by TLC and those with the pure product fraction were combined (CHCl 3 : MeOH =10:1 (vol/vol), R f (TA-PEG 600 -N 3 ) = 0.52, R f (TA) = 0.6, R f (N 3 -PEG 600 -NH 2 ) = 0.04). The solvent was evaporated to dryness, giving a yellow product, weight 2.3g, yield: 28%. 1  with CHCl 3 (200 μl × 5). The combined CHCl 3 layer was evaporated to dryness on a rotatory evaportor to yield a clear oil. 100 μl Et 3 N was then added to the product and mixed for 5 min, after which it was evaporated on a rotary evaporator. QD 600 (40 nmol/ml in tolune, 100 μl, 4 nmol) was repeatedly precipitate, centrifuged and washed with EtOH (500 μl × 4). The final QD residue was dissolved in CHCl 3 (500 μl) and mixed with the DHLA-PEG 600 -N 3 in a 20 ml round bottom flask under N 2 atmosphere at room temperature for 2 hrs. The solvent was then evaporated and residue was dissolved in EtOH. The resulting mixture was then transferred to 30,000 MW cutoff membrane and centrifuged at 7,000 × g for 3 mins followed by repeated washing with EtOH. The process was repeated three times to remove any unreacted DHLA-PEG 600 -N 3 . The final QD which has an effective MW > 30,000 and remained in the centrifuge tube was dissolved in EtOH. The quantum yield (QY) of the prepared QD-DHLA-PEG 600 -N 3 was found to be~6% using Rhodamine 6G (95% in ethanol) was the standard. Step i) Synthesis of 8,8-dibromobicyclo[5.0.1]octane. [2] Bromoform (2.45 mL, 28.5 mmol) was added dropwisely to a vigorously stirred creamy yellow mixture of cycloheptene (1.83 g, 19 mmol) and potassium tert-butoxide (4.26 g, 38 mmol) in anhydrous pentane (5 mL) at 0°C (ice-bath) under N 2 . The resulting brown mixture was then allowed to warm to room temperature and stirred overnight. Water (25 mL) was then added, and the reaction mixture was then neutralized with 1 M HCl. It was then transferred to a separation funnel and the aqueous phase was extracted with CHCl 3 (20 mL × 3). The combined organic phases were washed with water (20 ml × 3), dried on MgSO 4 , and then concentrated in vacco. The resulting yellowish-orange oil is purified by filtration on a short silica column, eluting with hexane to afford the desired product as a colorless oil (4.1 g, 90%) . 1  Step ii) Synthesis of methyl 2-bromocyclooct-1-en-3-glycolate. [2] Silver perchlorate (3.85 g, 18.6 mmol) was added to a solution of 8,8-dibromobicyclo[5.0.1]octane (2.5 g, 9.3 mmol) and methyl glycolate (6.35 mL, 83.9 mmol) in anhydrous toluene (5 mL) under N 2 and protected from light by an aluminium foil. The mixture was stirred at room temperature for 1.5 hrs. Silver salts are then filtered off and washed with EtOAc. The filtrate was concentrated and the residue was purified by column chromatography (10-20% EtOAc/hexane) to afford methyl 2-bromocyclooct-1-en-3-glycolate as a yellow oil (1.73 g, 67%). 1  Step iii) Synthesis of cyclooct-1-yn-3-glycolic acid. [2] Methyl 2-bromocyclooct-1-en-3-glycolate (816 mg, 2.94 mmol) was dissolved in anhydrous DMSO (8 mL) and heated to 60°C. 1,8-Diazabicyclo [5.4.0]undec-7-ene (DBU, 7mL) was added, the resulting mixture was stirred at for 15 min and more DBU (7mL) was added. The mixture was stirred at 60°C overnight and then cooled to room temperature. Water (20 mL) was added and solution was acidified to pH 1 with concentrated HCl. The aqueous phase was extracted with CHCl 3 (30 mL × 3). The combined organic layers were washed with saturated NaCl solution (30 mL), dried over MgSO 4 and filtered. The solvent was evaporated under reduced pressure and the crude product was purified by silica gel column chromatography (10:90:1-25:75:1 EtOAc:hexane:AcOH) to yield the product as a slight yellow solid (438 mg, 82%). 1

Step iv) Synthesis of cyclooct-1-yn-3-glycolic-N-hydroxysuccinimide ester (OCT-NHS)
Cyclooct Step v) Synthesis TBA-OCT 90 nmol TBA-NH 2 was dissolved in 90 µl water and then 210 µl ethanol was added to form TBA-NH 2 stock solution (300 µM). TBA-NH 2 (300 µM, 200 µl), OCT-NHS ester (0.312M,~200 µl), NaHCO 3 solution (50 mM, pH = 9, 20 µl) were mixed together (OCT-NHS:H 2 N-TBA molar ratio~1000:1). The resulting solution was kept at Step vi/vii) QD-TBA conjugation via copper free click chemistry TBA-Oct (~30 nmol, 90 μl) was mixed QD-DHLA-PEG600-N 3 (27.87 μM, 36 μl, QY~6.0%) in a fridge over night, leading to covalent QD-TBA conjugation via the CFCC. After that, OCT-COOH (0.1M, 10 μl) was then added to the mixture to quench any unreacted azide groups on the QD. This quenching step was found to be important in terms of improving the stability of the QD-TBA n conjugate. The mixture was allowed to stand at room temperature for a further two hours. The reaction mixture was transferred to a centrifugation vial equipped with a 30,000 WMCO cutoff membrane and centrifuged at 20,000 × g followed by washing with water. An absorption spectrum was taken on the combined clear centrifuge/wash mixture and the absorbance at 260 nm was used to calculate the amount of TBAs not conjugated to the QD. This gave a value of~10 nmol. Therefore~20 nmol of TBAs have conjugated to the QD-DHLA-PEG600-N 3 (1 nmol), giving the copy number of TBAs attached to each QD of~20, denoted as QD-TBA20. The numbers of TBA strands conjugated to each QD varied from batch to batch, but were generally in the range of 15~20. The final QD-TBA 20 was dissolved in 7 μM BSA aqueous solution for long term storage. Its quantum yield (QY) was determined using Rhodamine 6G in ethanol as the standard using the equation below: Where QY QD , F QD and A QD are the QY, integrated fluorescence intensity and absorbance at 480 nm of the QD sample, and QY S , F S and A S are the quantum yield, integrated fluorescence intensity and absorbance at 480 nm of the standard (Rhodamine 6G, which has a QY of 95% under 480 nm exitation). The QY of the QD-TBA 20 was found to be~5.9%, effectively the same as that of the parent QD-DHLA-PEG600-N 3 (~6.0%). [4] Step 1) Preparation of glutathione modified QD Glutathione capped QD was prepared by following our previously established procedures. [4] Briefly, TOPO-QD (80 nmol) in toluene were precipitated by EtOH. The precipitate was dissolved in chloroform (1 ml), into which was added 200μl GSH solution containing 28.4 mg GSH and 20 mg KOH in MeOH. The mixture was shaken for 30 min, and a precipitate was formed. After centrifugation, the clear supernatant was discarded and the precipitate was washed with MeOH 3 times to remove any uncapped GSH. The precipitate was dissolved in pure water to form a bright red stock solution and stored in a fridge at 4 o C till use, denoted as QD-GSH. Its concentration was determined from its absorbance at the exciton peak by using the Beer-Lambert law following the previously established method. Its QY was estimated as~18% using Rhodamine 6G in ethanol (QY = 95%) as standard.

Preparation of covalently coupled QD-DNA conjugate
QD-DNA conjugation was performed by following our previous procedures. [3] Briefly, EDC (30 mg) and NHS (15 mg) were dissolved in 200 μL phosphate buffer (20 mM, pH = 5.57) into which was added 2.0 nmole of QD-GSH (223 μL, 8.98 μM), and the resulting solution was sonicated for 20 s. The mixture was allowed to stand at room temperature for 1 h to activate the carboxylic acid groups in QD-GSH into active NHS esters. After that, the solution was centrifuged (at 21,000 × g for 3 mins), and the clear supernatant was discarded. The precipitate pellet was washed carefully with water (2 × 100 μL). After that, the QD pellet was added with H 2 N-TBA (200 μM 150 S8 μL, 30 nmole), and NaHCO 3 buffer (50 mM, pH = 9, 100 μL) and thoroughly mixed, sonicated and then was allowed to stand overnight at 4℃. The resulting solution was then added EtOH (400 uL) and stored in a freezer for 1 h. After that, the solution was centrifuged and the clear supernatant which contained the unreacted TBA was carefully removed (checked with an UV lamp to ensure no QD was removed). The pellet was carefully washed with a mixed solution of EtOH (400 uL), water (100 uL) and 50 mM NaHCO 3 (100 uL). An absorption spectrum was taken on the combined clear supernatant/wash and the absorbance at 260 nm was used to calculate the amount of TBA not conjugated to the QD, giving a value of~20 nmol. Therefore 10 nmol of the TBA were conjugated to the QD-GSH (2 nmol), so the number of TBA strands attached to each QD was 5, abbreviated as QD-TBA 5 .
Finally, the pellet was dissolved in 0.5 mL of pure water to obtain a clear QD-TBA 5 stock solution and stored in the dark at 4°C till use.

C4) Hybridization of probe DNAs to the QD-TBA 20 .
The total volume of the hybridization solution was 400 L, contianing a final QD concentration of 2 nM in 1× PBS (10 mM phosphate, 150 mM NaCl, pH 7.40). The hybridization reaction was carried out in batches under identical conditions, where 40 L of the 10 × PBS, BSA and Cys-His 6 peptide, DNA probe, and the required amount of the QD-TBA 20 and the required amount of water were added to a series of eppendorfs, and thoroughly mixed. The hybridization reaction was carried out at room temperature for 2 hrs before fluorescence spectra were taken. All fluorescence spectra were recorded on a Fluoromax-3 fluorescence spectrophotometer. The emission spectra (550 -750 nm) were recorded under a fixed excitation wavelength of 450 nm ( abs minimium of the Atto647N acceptor to minimize its direct excitation) at a scan rate of 120 nm/min. Excitation and emission bandwidths of 5 nm were used.
The quantum yield of the QD was measured using Rhodamine-6G in ethanol (95% under 480 nm excitation) as a reference. The optical densities of the QD and Rhodamine-6G solutions used were 0.05 at 480 nm. C5) Calculation of FRET signal. [3] The fluorescence intensities, I QD and I Dye , in this paper are the integrated fluorescence, while I 605 and I 670 are the fluorescence intensities at 605 and 670 nm (corresponding to the  EM maximum of the QD and dye respectively).
The Atto647N FRET signal was obtained by subtracting the QD fluorescence from the total integrated fluorescence of the whole spectrum (after correction for direct excitation of Atto647N). The QD fluorescenec was calculated assuming that the QD fluorescence maintained the same shape as the QD-TBA 20 only sample, so its integrated fluorescence is proportional to the height of the QD fluorescence peak at 605 nm where Atto647N does not fluoresce at this wavelength.   Soc. 2004, 126, 301), which in term leads to a greater R 0 value and hence an improved FRET ratio.

Estimation of the amount of QD adsorption
The possible maximum amount of QD adsorbed on surfaces can be simply estimated as follows: 1) assuming the QD-TBA 20 s are rigid balls with a diameter of 10 nm and are closely-packed when they are adsorbed on the surface, then each QD would occupy a surface area = 3.14 × (10/2) 2 = 86.6 nm 2 or 8.66 ×10 -13 cm 2 ; 2) 1 mL of the QD sample is stored in 1 mL (1cm × 1cm × 1cm) cuvette, this would give a possible surface contact area of 5 cm 2 (the top surface is ignored). Under such assumptions, then the number of surface adsorbed QDs = 5/[8.66×10 -13 ] = 5.77 × 10 12 , which is equivalent to 9.6 pmole (5.77×10 12 /(6.02 × 10 23 ) or 9.6 nM in 1 mL sample. Therefore, it is entirely possible that all of the QDs can be completely adsorbed onto the surface when a low concetrantion of 2 nM was used in this paper. respectively. The I 605 was found to decrease slightly while the I 665 FRET signal increased slightly, leading to an increased I 665 /I 605 ratio as the hybridization time was increased from 1 to 18 hr. The increase of the I 665 /I 605 ratio was minimum at low DNA: QD ratio (≤ 15), but become more apparent as the DNA29:QD ratio was increased to > 20:1, suggesting that it may take some time for the DNA29 to fully hybridize to all of the available the QD