Quantum dots as labels in a hairpin DNA sensor

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Chapter 2 – Materials and methodology

This chapter describes the experimental details and the methodologies used in this work. As the techniques, which have been utilized for characterization purposes or to detect the sensor response, are extensively described in the literature only the basic principles for each of the techniques are presented here.


Quantum Dots (QDs)
CdSe/ZnS QDs

The synthesis of CdSe/ZnS core/shell QDs was done according to the literature.57, 59 Briefly, CdO was added to mixture of n-dodechylphosphonic acid, trioctylphosphine oxide (TOPO) hexadecylamine (HDA), degassed and heated to 300 °C under argon flow, followed by stabilization at 270 °C. A solution of Se in trioctylphosphine (TOP) was quickly injected to the mixture. The resulting CdSe nanocrystals were redispersed in heptane with subsequent addition of TOPO and HDA, followed by heating to 200 °C under argon flow. A mixture of zinc ethylxanthate in dioctylamine, and zinc stearate dispersed in octadecene, was injected at a rate of 4.8 ml h-1 to create the ZnS shell around the core crystals.

CdTe QDs

The synthesis of CdTe QDs was performed according to the procedure developed by Rogach et al.,66 with some modifications. First, NaHTe was prepared by adding 40 mg NaBH4 to a flask containing 46 mg tellurium powder and 2 ml Milli-Q water under nitrogen atmosphere. The reaction was kept on for several hours until all tellurium powder was dissolved. 0.092 g (0.5 mmol) of CdCl2 and 0.092 mg (1 mmol) of thioglycolic acid were dissolved in 100 ml Milli-Q water, followed by adjusting pH to 8.2 by addition of 1 M NaOH solution. The mixture was deaerated by N2 bubbling for 30 min. Then NaTeH solution (0.062 mmol) was quickly injected into the mixture under vigorous stirring, followed by refluxing the mixture for 10 min under open-air conditions. 400 ml of 2-propanol was added to as-prepared CdTe QDs colloid solution to precipitate the CdTe QDs , which were collected by centrifugation. The obtained CdTe QDs were dried at room temperature under vacuum, dissolved in 10 ml of Milli-Q water and then used as prepared for the subsequent experiments.

Ligands for functionalization of the CdSe/ZnS QDs

Ligand 1 (8-thio-3,6-dioxaoctanol) and 2 (DSBA, (5-(6,8-diaza-7-oxo-3-thiabicyclosec[3.3.0]oct-2-yl)-N[7-(3-{[2-(N{7-[5-(6,8-diaza-7-oxo-3 thiabicyclo[3.3.0]oct-2-yl)pentanoylamino]heptyl}mcarbamoyl)ethyl]disulfonyl}propanoylamino)heptyl]pentan amide)), which were used for the functionalization of the CdSe/ZnS QDs, were synthesized according to Charvet et al.57, with some modifications, following the pathways visualized in Schemes 2.1 and 2.2, respectively.

Ligand 1 (8-thio-3,6-dioxaoctanol)

Ligand 1 was obtained by dissolving 29.6 mmol of 2-(2-(2-chloroethoxy)ethoxy)ethanol and 39 mmol of thiourea in 15 ml of milli-Q water and stirring under reflux for 2.5 h. A solution of 2.5 g sodium hydroxide dissolved in 20 ml of milli-Q water was added and the reflux was continued at 95 °C for another 2.5 h. After cooling the mixture, 6 ml of concentrated sulfuric acid in 4 ml of milli-Q water was used for acidification and 5×25 ml of ethyl acetate was used for the extraction of the aqueous phase. The collected organic phases were dried over sodium sulfate, filtered, evaporated under reduced pressure and purified on silica (chloroform/methanol 8:1 and ethylacetate/hexane 2:1) to yield Ligand 1, as a colourless oil.67

Ligand 2 (DSBA)

Ligand 2 was synthesized from the precursors A, B and C (biotin N-hydroxysuccinimide ester, N-(13-amino-4,7,10-trioatridecanyl) biotinamide and 11-mercaptoundecanoyl-N-hydroxysuccinimide ester, respectively) as shown in Scheme 2.2

Biotin N-hydroxysuccinimide ester, compound A

Biotin N-hydroxysuccinimide ester was synthesized by adding 24.44 mmol of 1,3-dicyclohexylcarbodiimide to a stirred solution of equimolar amounts (20.47 mmol) of biotin and N-hydroxysuccinimide in 150 ml of anhydrous N,N-dimethylformamide (DMF) according to Bayer et al.68 The mixture was stirred at room temperature for 48 h. The formed dicyclohexylurea was filtered off and the solvent was evaporated under reflux. 500 ml of diethyl ether was added to the residue and the solution was stirred for 2 h to yield a white precipitate, which was filtered under reduced pressure and recrystallized in isopropanol. The final product, 4.2 g of a white powder, was allowed to dry in a vacuum oven at 50 °C for 3 – 4 hours before further use.

N-(13-Amino-4,7,10-trioxatridecanyl) biotinamide, compound B

The procedure on preparation of N-(13-Amino-4,7,10-trioxatridecanyl) biotinamide was reported by Wilbur et al.69 4.2 g of biotin N-hydroxysuccinimide ester, A, was dissolved in 100 ml of dry DMF. Under inert atmosphere, this solution was added dropwise within 1 h to a solution of 13.56 g of 4,7,10-trioxa-1,13-tridecanediamine in 4 mL of triethylamine. The reaction mixture was stirred at room temperature for 65 h, after which the solid that was formed was filtered off. Evaporation of DMF from the solid was carried out under reduced pressure. The resulting oil was added dropwise, and under continuous stirring, to 850 ml of hexane and a white precipitate was formed. The solution was stirred for several hours followed by recrystallization in isopropanol for 2 h at reflux and then for 12 h without heating or stirring. After drying the product in a vacuum oven at 50 °C a final yield of 5.7 g of white crystals was obtained.


mercaptoundecanoyl-N-hydroxysuccinimide ester, compound C

Connolly et al.70 has published the preparation procedure for 11-mercaptoundecanoyl-N-hydroxysuccinimide ester as follows: 1.04 g of N-hydroxysuccinimide in 500 ml of dichloromethane was stirred for 30 min followed by addition of 1.88 g of 11-mercaptoundecanoic acid (MUA), dissolved in 10 ml of dichloromethane. 1.95 g of 1,3-dicyclohexylcarbodiimide was dissolved in 50 ml of dichloromethane and added dropwise, within 30 min, to the reaction mixture. The mixture was then stirred for 26 h and after filtration and evaporation of the solvent the residue was purified with flash chromatography (hexane/diethylether, 1:1) and approximately 1.2 g of 11-mercaptoundecanoyl-N-hydroxysuccinimide ester was obtained.

Ligand 2

Once 11-mercaptoundecanoyl-N-hydroxysuccinimide ester, C, is obtained it can be reacted with N-(13-amino-4,7,10-trioxatridecanyl) biotinamide, B, to form ligand 2.70 894 mg of B was added to a solution of 630 mg C, in 100 ml of dry chloroform and 200 ml of triethylamine. The reaction mixture was stirred at room temperature for 4 h under inert atmosphere. Chloroform was evaporated under reduced pressure and the residue was purified by flash chromatography (dichloromethane/methanol, 9:1, ethylacetate/methanol 20:1, ethylacetate/methanol 5:1 and methanol). 1.1 g of yellowish oil was obtained as the final product.7

Functionalization of CdSe/ZnS QDs

Ligand 1 and 2
In order to obtain water-soluble CdSe/ZnS QDs, ligands 1 and 2 were used for functionalization (biotinylation) of the synthesized QDs. This was done according to the procedure described by Charvet et al.,57 with some modifications.
21.7 mg of ligand 1 and 4.9 mg of ligand 2 dispersed in 20 µl of dry chloroform was mixed with 500 µl of a 8 g l-1 QD-solution. The mixture was stirred under inert atmosphere, at room temperature and covered to prevent photodegradation for 48 h. Then 500 µl of milli-Q water and 100 µl of acetone were added and the mixture was stirred for 1 h. Another 100 µl of water was added and the stirring continued for another 10 min. After separation of the aqueous layer the solution was centrifuged at 1320 rpm for 5 min. No precipitate was obtained and thus the amount of solvent was reduced by heating the solution under nitrogen flow. Again, 100 µl of water was added to the sample, followed by centrifugation. This procedure was repeated two times before the QD precipitated. The supernatant was removed, followed by evaporation to dryness to yield a colored solid, which was redispersed in water for further use.

Thioglycolic- and dihydrolipoic acid (TGA and DHLA)

Thioglycolic acid (TGA) and dihydrolipoic acid (DHLA) were also used for the replacement of the TOPO-ligands on the hydrophobic QDs. The procedure reported by Clapp et al.71 was followed with some modifications. 100 µl of 0.28 M DHLA or 1 ml of 1 M TGA was mixed together with 200 µl of 0.26 M CdSe/ZnS QDs and added to 1 ml of milli-Q water. A further 800 µl of chloroform was added and the mixture was shaken at 1000 rpm at room temperature for 18 h (DHLA) or 90 h (TGA). After it could be visibly established that the QDs had transferred to the aqueous phase the chloroform was removed. The remaining suspension was centrifuged through a micro centrifuge filter, with a nominal molecular weight limit of 100 000 Da, at 2200 rpm for 4 min and used as prepared.

Materials and Chemicals
Oligonucleotides (ODNs)

The DNA oligonucleotide (ODN) sequences were synthesized by Alpha DNA (Quebec, Canada) and the sequences used in the sensor designs are listed in Table 2.1. Table 2.2 contains additional sequences, used for characterization of the HPP and its duplex formation with different targets in solution (Section 3.3)

Chapter 1 – General introduction
1.1 Objectives
1.2 Deoxyribonucleic acid (DNA)
1.3 DNA sensors
1.4 Self-assembled monolayers (SAMs) on gold
1.5 Quantum Dots (QDs)
Chapter 2 – Materials and methodology 
2.1 Synthesis
2.2 Materials and Chemicals
2.3 Formation of self-assembled monolayers (SAMs) as sensor platforms
2.4 Characterization techniques
Chapter 3 – Characterization of the components of the general DNA sensor design
3.1 Investigation of the quenching ability of Au and monolayered m-PEG of the fluorescence of Cy3 dye and CdSe/ZnS quantum dots
3.2 Characterization of the mixed SAMs
3.3 Characterization of the HPPs
Chapter 4 – Neutron Reflectometry Study of a Poly(ethylene glycol ) and Hairpin Probe Self-Assembled Monolayer used for DNA-sensing
4.1 Introduction
4.2 Neutron Reflectometry analysis
4.3 Characterization of mSAMs before and after hybridization with complementary target
4.4 Polarized neutron reflectometry for investigation of the melting behavior of the surfaceattached probes in the mSAM
4.5 Conclusions
Chapter 5 – Quantum dots as labels in a hairpin DNA sensor 
5.1. Introduction
5.2 The optical CdSe/ZnS-labeled hairpin sensor
5.3 The electrochemical CdTe-labeled hairpin sensor
5.4 Conclusions
Chapter 6 – Effect of Probe Density and Hybridization Temperature on the Response of an Electrochemical Hairpin-DNA Sensor 
6.1 Immobilization of HPP and m-PEG molecules onto a gold electrode
6.3 Hybridization dynamics and sensitivity of the label-free DNA sensor
6.4. Selectivity of the label-free DNA sensor
6.5. Conclusions
Chapter 7 – Summary and future work 
7.1 Summary
7.1 Future work

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