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Biological and Medical Applications of Materials and Interfaces

Sub-nanomolar FRET-based DNA Assay Using Thermally Stable Phosphorothioated DNA-functionalized Quantum Dots Jae Chul Park, Se Yeon Choi, Moon Young Yang, Lin Nan, Hyebin Na, Ha Neul Lee, Hyun Jung Chung, Cheol Am Hong, and Yoon Sung Nam ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b07717 • Publication Date (Web): 28 Aug 2019 Downloaded from pubs.acs.org on August 30, 2019

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ACS Applied Materials & Interfaces

Sub-nanomolar FRET-based DNA Assay Using Thermally Stable Phosphorothioated DNA-functionalized Quantum Dots Jae Chul Park1, Se Yeon Choi1, Moon Young Yang2, Lin Nan1, Hyebin Na1, Ha Neul Lee3, Hyun Jung Chung3,4, Cheol Am Hong5,*, and Yoon Sung Nam1,2,* 1

Department of Materials Science and Engineering, 2KAIST Institute for NanoCentury, Graduate School of Nanoscience and Technology, and 4Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea 3

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School of Chemistry and Biochemistry, Yeungnam University, 280 Daehak-Ro, Gyeongsan, Gyeongbuk, 38541, Republic of Korea

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ABSTRACT Quantum dots (QDs) can serve as an attractive Förster resonance energy transfer (FRET) donor for DNA assay due to their excellent optical properties. However, the specificity and sensitivity of QD-based FRET analysis are prominently reduced by non-specific DNA adsorption and poor colloidal stability during DNA hybridization, which hinders the practical applications of QDs as a bio-sensing platform. Here we report sub-nanomolar FRET assay of DNA through the stabilization of DNA/QD interface using DNA-functionalized QDs with phosphorothioated single-stranded DNA (pt-ssDNA) as a multivalent ligand in an aqueous solution. In situ DNA functionalization was achieved during the aqueous synthesis of CdTe/CdS QDs, resulting in the maximum photoluminescence quantum yields of 76.9 % at an emission wavelength of 732 nm. Conventional monothiolated ssDNA-capped QDs exhibited particle aggregation and photoluminescence (PL) quenching during DNA hybridization at 70 oC due to the dissociation of surface ligands. Such colloidal instability induced the non-specific adsorption of DNA, resulting in false-positive signal and decreased sensitivity with the limit of detection (LOD) of 16.1 nM. In contrast, the pt-ssDNAfunctionalized QDs maintained their colloidal stability and PL properties at the elevated temperature. The LOD of the pt-ssDNA-functionalized QDs was > 30 times lower (0.47 nM) while maintaining the high specificity to a target sequence because the strong multivalent binding of ptssDNA to the surface of QDs prevents the detachment of pt-ssDNA and non-specific adsorption of DNA. The study suggests that the ligand design to stabilize the surface of QDs in an aqueous milieu is critically important for the high performance of QDs for specific DNA assay. KEYWORDS: Förster resonance energy transfer; phosphorothioated DNA; quantum dots; DNA analysis; colloidal stability


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INTRODUCTION Quantitative analysis of nucleic acids has proven to be crucial in the fields of food safety,1 environmental monitoring,2 and early diagnosis of diseases.3-5 Various techniques for the sensitive detection of specific nucleic acids have been developed, such as polymerase chain reaction (PCR)based techniques,6,7 fluorescence-based assays,8 electrochemical sensing,9 and surface-enhanced Raman scattering.10 Despite the high sensitivity, these methods have limitations for practical applications because of high cost, long processing time, and complexity.11 Förster resonance energy transfer (FRET)-based sensing can be used as an excellent sensing platform for simple, fast, and sensitive DNA detection.11-13 Since the sensitivity of a FRETbased sensor relies on energy transfer between donor and acceptor fluorophores, the choice of proper FRET pairs is critically important. Quantum dots (QDs) have been considered as an efficient FRET donor compared to organic fluorophores because of their large absorption crosssection, broad absorption spectra, tunable emission wavelength, narrow emission bandwidth, high photoluminescence quantum yield (PLQY), and excellent photostability.14-24 Because of their promising properties, many studies reported hybrid systems composed of QDs and DNA, enabling the detection of various nucleic acid biomarkers.25-28 Despite the great promise of QDs as a FRET donor, there remain several issues that should be resolved before the practical applications of QDs to DNA assay. The thermal stability, which indicates the colloidal and PL stabilities at an elevated temperature, of DNA-functionalized QDs is particularly required when the hybridization process involves heating to an elevated temperature, usually in the range of 65-90 oC. Although it is possible to hybridize short oligonucleotides at room temperature, heating is essentially required to ensure efficient DNA annealing by denaturing the secondary structure, heterodimer, and self-dimer of oligonucleotides.29-32 However, the thermal


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stability of DNA-QDs has not yet been well studied though it has been known that a thiol moiety, the most commonly used binding moiety for QDs, cannot prevent PL quenching at 75 oC.33 PL quenching occurs when the binding affinity of surface ligands is insufficient to overcome thermal fluctuation, which induces the dissociation of ligands and aggregation-induced PL quenching. Therefore, the surface stabilization of QDs with DNA is required to maintain colloidal and PL stability at an elevated temperature. Several conjugation strategies have been introduced for the DNA functionalization of QDs using thiol-modified single-stranded DNA (ssDNA) and streptavidin-biotin interactions.34-37 However, thiol-moiety cannot provide sufficient thermal stability, and the relatively large size of streptavidin (ca. 5 nm)38,39 significantly increases the overall distance between FRET pairs, lowering FRET efficiency and the sensing performance. In addition, these methods often involve the phase transfer of oil-dispersible QDs to an aqueous solution before DNA functionalization, significantly reducing their PLQY.40-43 Recently, in situ DNA functionalization during the aqueous synthesis of QDs was developed using phosphorothioated ssDNA (pt-ssDNA) to achieve a high PLQY.44-50 Phosphorothioate is a sulfur-containing variant of the phosphodiester backbone, so it can preferentially bind to the metal elements of QDs. Because pt-ssDNA can increase the binding affinity by increasing the number of phosphorothioate-modified bases,45,46 we hypothesized that pt-ssDNA-functionalization can be an excellent solution to achieve the high thermal stability of QDs for DNA hybridization. However, the application of pt-DNA-functionalized QDs to DNA assay with an investigation on the thermal stability has not yet been reported. In this study, we demonstrated the importance of ligand stabilization on the surface of QDs in an aqueous milieu for high FRET performance for DNA assay. To this end, we synthesized DNA-functionalized, highly luminescent CdTe/CdS QDs through in situ pt-ssDNA


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functionalization, denoted by ‘ptDNA-QDs,’ and investigated their thermal stability and FRET performance for DNA assay. In particular, the thermal stability of ptDNA-QDs was investigated by measuring the PL spectra and hydrodynamic diameters before and after the exposure to the DNA annealing condition, which includes heating to 70 oC, followed by cooling to ambient temperature. FRET analysis was conducted to examine the effects of thermal stability of the DNAfunctionalized QDs on FRET-based DNA analysis. Monothiolated ssDNA-functionalized QDs, denoted by ‘mtDNA-QDs,’ were also used as a control for comparison in terms of sensitivity and target specificity. Finally, the limit of detection (LOD) of FRET assays was determined using ptDNA-QDs and non-fluorescent BHQ2 DNA (Black Hole Quencher®-2) as the FRET donor and acceptor, respectively.

EXPERIMENTAL SECTION

Chemicals and Materials. Tellurium powder (Te, < 200 mesh, trace metals basis, 99.8 %), sodium borohydride (NaBH4, ≥ 98 %), 3-mercaptopropionic acid (MPA, ≥ 99 %), cadmium nitrate tetrahydrate (Cd(NO3)2∙4H2O), ≥99 %), rhodamine 6G, and 10 M sodium hydroxide (NaOH, Bio-ultra grade), and tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 0.5 M, pH 7.0) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethanol (99.9 %) was purchased from Daejung Chemicals & Metals Co., Ltd (Siheung, Republic of Korea). UltrapureTM diethylpyrocarbonate (DEPC)-treated water was purchased from Thermo-Fisher (Waltham, MA, USA). Milli-Q water with a conductivity of 18.2 MΩ was used to prepare all aqueous solutions except for the DNA-related reaction, where the DEPC-treated water was used. All ssDNA oligonucleotides were synthesized by Bioneer, Co. (Daejeon, Republic of Korea). The sequences of the oligonucleotides are as follows: target DNA, 5’- TATCT TGTAT TTATC GATTT AGTAT


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TTTCA TGAAT CCA -3’; non-complementary DNA, 5’-TGGAT TCATG AAAAT ACTAA ATCGA TAAAT ACAAG ATA-3’; hairpin target DNA, 5’-TATCT TGTAT TTATC GATTT AGT AT TTTCA TGAAT CCATG GATTC ATGAA AATAC TAAAT CGATA AATAC AAGAT A-3’; Cy5-labeled DNA (Cy5-DNA), 5’-Cy5-AATCG ATAAA TACAA GATA-3’; BHQ2-labeled DNA (BHQ2-DNA), 5’-BHQ2-AATCG ATAAA TACAA GATA-3’; pt-ssDNA, 5’-(A*)7-AAAAA TGGAT TCATG AAAAT ACTA-3,’ where A* denotes adenine modified with the phosphorothioate backbone; and mt-ssDNA, 5’ thiol-AAAAA TGGAT TCATG AAAAT ACTA3’. The sequences of the oligonucleotides for detecting Klebsiella pneumoniae carbapenemase (KPC) are as follows: target DNA, 5’- TGGAC ACACC CATCC GTTAC GGCAA AAATG CGCTG GTTC CGTGG TCACC CATCT C-3’; BHQ2-DNA, 5’-BHQ2-CCAGC GCATT TTTGC CGTA-3’; and pt-ssDNA, 5’-(A*)7-AAAAA GAGAT GGGTG ACCAC GGAA-3.’ Synthesis of CdTe Core QDs. Briefly, 127.5 mg (1.0 mmol) of Te powder and 112.6 mg (3 mmol) of NaBH4 were mixed with 2.0 mL of Ar-saturated deionized water to prepare a NaHTe solution. Ar blowing with a small outlet was maintained to prevent the oxidation. After 2 h, a transparent purple solution with white precipitates was obtained. In the meantime, a cadmium precursor solution was prepared by dissolving 77.12 mg (0.25 mmol) of Cd(NO3)2 and 50 μL of MPA in 50 mL of deionized water under vigorous stirring. The pH of the solution was then adjusted to 12.2 by dropwise adding 1 M NaOH. The solution initially became turbid and then transparent at the final pH. Then, the solution was degassed under vacuum for 30 min and then bubbled with Ar for 30 min. A drop of the NaHTe solution (about 20 μL) was then injected through a syringe with a 21G needle into the Cd precursor solution under Ar-bubbling. The CdTe core QDs were synthesized and confirmed by the color change from transparent to faint yellow. The molar ratio of Cd/MPA/Te was fixed to 1:2.23:0.04. The high ratio of Cd/Te facilitates the growth of magic-


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sized CdTe clusters. The solution was aged at 4 oC until the emission peak reached 490 nm. These core QDs were purified by the addition of ethanol (25 vol%), followed by centrifugation at 6,500 rcf for 15 min. The precipitated QDs were subsequently re-dispersed in DEPC-treated water to recover the initial volume. Synthesis of DNA-functionalized CdTe/CdS Core/Shell QDs. To prepare ptDNA-QDs, we diluted the concentration of re-dispersed CdTe core QD suspension to 120 nM. The concentration of the CdTe QDs was calculated following a reported method.51 Then we mixed 1 mL of the diluted QD solution with 45 μL of 25 mM Cd2+ and 90 μL of 25 mM MPA, followed by sonication for 1 min. Next, 0.5 mL of 20 μM phosphorothioate DNA was added and gently vortexed. The pH was adjusted to 12.2 by adding 1 M NaOH. The reaction mixture was placed in a water bath and heated at 90 oC for 40 min and then cooled down by immersing the vial in a water bath at room temperature. Lastly, 0.5 mL of the reacted solution was moved into a 0.5 mL Amicon filter (MWCO 30KDa) and centrifuged at 4,602 rcf for 3 min. The concentrated solution was mixed with 350 μL of DEPC-treated water and centrifuged at 4,602 rcf for 3 min. These procedures were repeated three times. The synthesis of mtDNA QDs was modified from the synthetic procedures of ptDNA-QD. The re-dispersed CdTe core QDs were diluted twenty times in 1 mL of DEPC-treated water as described above in the synthetic part of ptDNA-QD. Next, 1 mL of the diluted QD solution was mixed with 45 μL of 25 mM Cd2+ and 90 μL of 25 mM MPA, followed by sonication for 1 min. For DNA-functionalization, 0.5 mL of 200 nM thiol-ssDNA solution and 2 μL of 0.5 M TECP solution were added and gently vortexed. The pH was adjusted to 12.2 by adding 1 M NaOH. The reaction mixture was placed in a water bath and heated at 90 oC for 40 min and then cooled down by immersing the vial in a water bath at room temperature. The mtDNA-QD solution was purified


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four times with a 0.5 mL Amicon filter (MWCO 30KDa), which is the same as for the ptDNAQDs described above. FRET Analysis. For FRET analysis, DNA-QD with an emission peak of about 620 nm was used as a FRET donor, and BHQ2 (or Cy5) was used as a FRET acceptor. FRET assay was carried out using 20 nM of DNA-QDs and 500 nM of BHQ2-DNA (or Cy5-DNA) mixed in DEPCtreated water. For LOD measurement, target DNA from 0 to 500 nM was added. For DNA hybridization, DNA annealing was conducted by heating at 70 oC for 10 min, followed by incubation at room temperature for 10 min. The DNA concentration was quantified using a UVvis spectrophotometer (ND-1000, NanoDrop Technologies, Wilmington, USA). PL spectra were obtained using an F-7000 fluorescence spectrophotometer (Hitachi High-technologies, Tokyo, Japan). The CLARIOstar® microplate reader (BMG Labtech, Aylesbury, United Kingdom) was also used for LOD measurement to get a calibration curve, and the fluorescence intensity of the blank sample was measured for twenty times. Cy5-DNA was used instead of BHQ2-DNA, and FRET assays were performed in a black 96-well microtiter plate with a working volume of 300 μL with the excitation at 400 nm. Characterization. Absorption spectra were measured with a UV-vis spectrophotometer (UV-1800, Dong-il Shimadzu Corporation, Seoul, Republic of Korea). PL spectra were measured with an F-7000 fluorescence spectrophotometer. The PLQY of the QDs was measured using both absolute and relative methods. Absolute PLQY was measured with a Quantum Efficiency Measurement System QE-2000 (Otsuka Electronics Co., Ltd, Osaka, Japan) equipped with an integrating sphere. Relative PLQY was measured using rhodamine 6G in ethanol as a reference dye.52 Transmission electron microscopy (TEM) was performed on a JEOL JEM-3010 (Tokyo, Japan) to characterize the size and structure of QDs. Time-resolved PL decay spectra were


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measured with excitation from a 478-nm pulsed diode laser using a fluorescence lifetime spectrometer (FL920, Edinburgh Instruments, Livingston, UK). The hydrodynamic diameter of QDs was measured using an ELSZ-2000 (Otsuka Electronics Co., Ltd, Osaka, Japan). Density functional theory calculations. Density functional theory (DFT) calculations were carried out on the basis of the revised Perdew-Burke-Enzerhof generalized gradient approximation (RPBE-GGA)53,54 implemented with projector augmented-wave (PAW) pseudopotential55 using the Vienna ab initio Simulation Package (VASP).56 The implicit solvation effect was applied by using VASPsol.57 The cutoff energy for basis functions was 400 eV, and a 2 × 2 × 1 Monkhorst-Pack grid was used for the Brillouin zone integration. All of the structures were relaxed until the forces on the atoms were less than 0.05 eV/Å, and periodic boundary conditions were applied to all three dimensions. The calculated lattice constant for CdS was a = b = c = 5.92 Å, which is slightly larger (1.7%) than the experimental data, 5.82 Å.58 The [100] facet of zinc blende CdS structure consisted of 40 atoms, where the bottom layer atoms of the CdS structure were fixed.

RESULTS AND DISCUSSION

The synthetic procedures for the DNA-functionalized CdTe/CdS QDs are schematically described in Figure 1. Typically, Cd(NO3)2 and MPA were mixed in deionized water in a three-neck flask, and the pH of the solution was adjusted to 12.2. At the high pH, highly soluble Cd-thiolate complexes are generated in a molecular state. When NaHTe was injected, the Cd-thiolates were decomposed to form CdTe monomers, resulting in the nucleation and growth of CdTe QDs.59 The synthesized QDs were aged at 4 oC until the emission peak reached 490 nm, followed by purification and re-dispersion in DEPC-treated water. For the CdS shell growth and in situ DNA


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functionalization, the pre-determined amounts of Cd-thiolates and pt-ssDNA were added to the CdTe QD solution, followed by adjusting the pH to 12.2 and heating at 90 oC. As the Cd-thiolate complexes were slowly decomposed, a CdS shell gradually passivated the CdTe QDs. During the shell growth, seven S- in the phosphorothioate-modified backbone were bound to the QDs. Five adenine nucleotides were added as a spacer to extend the targeting sequence away from the QD surface, enabling the hybridization with its complementary ssDNA.

Figure 1. Schematic illustration of the aqueous phase synthesis of CdTe/CdS QDs with in situ ptssDNA functionalization.

For the epitaxial growth of the CdS shell, the size of CdTe QDs should be very small to form a so-called ‘magic-size’ cluster.9 To synthesize the magic-size CdTe QDs, we adjusted the molar ratio of Cd to Te to 25:1.60,61 Transmission electron microscopy (TEM) reveals that the diameter of CdTe QDs is smaller than 2.5 nm (Figure 2a). The high-resolution TEM (HRTEM) image of CdTe QDs (inset of Figure 2a) shows clear lattice fringes with a d-spacing of 0.37 nm, corresponding to the (111) plane of the zinc blende CdTe. The normalized absorption (the local maximum peak at 437 nm) and PL spectra (the maximum peak at 495 nm) of the synthesized CdTe


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QDs also indicate that the synthesized CdTe QDs have a small diameter of about 2 nm (Figure 2b).46,59

Figure 2. TEM image (a) and absorption and steady-state PL spectra (b) of the synthesized CdTe core QDs. Inset of a: a high-resolution TEM image of the CdTe QDs. The d-spacing of CdTe QDs indicates the (111) plane of zinc blende structure of CdTe. The excitation wavelength was 400 nm for the PL measurement.

After the CdS shell growth, the size of QDs was increased as shown in Figure S1. The HRTEM image of CdTe/CdS QDs (inset of Figure 3a) shows a clear lattice fringe with a d-spacing of 0.21 nm, corresponding to the (220) plane of the zinc blende CdS. The results support that the CdS shell was successfully grown on the surface of the CdTe core. The normalized absorption and PL spectra of the CdTe/CdS QDs with different shell growth times are shown in Figure 3b. The absorption and PL maximum peaks were red-shifted when the shell growth time was increased, while the full width at half-maximum (FHWM) does not change as a function of the shell growth time (Figure 3c). Time-resolved PL decay curves are plotted in Figure 3d, and the average PL lifetimes, which were calculated by the amplitude-weighted average of the lifetime components from two-exponential fits, were increased from 36 ns to 100 ns with the increased shell growth time (Figure S2). These spectral changes are ascribed to the strain-induced transition from type-I to type-II structure, as discussed in Figure S3. The relative PLQY was also increased with the increased shell growth time, indicating that the CdS shell effectively passivated surface defects and suppressed non-radiative decay. The maximum PLQYs were 76.9 % and 70.2 % for relative


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and absolute analyses, respectively, when QD has an emission peak of 732 nm. The error between the relative PLQY and absolute PLQY would be originated from measuring the relative PLQY of NIR-emitting QDs with rhodamine 6G. Because the magic-size QDs have a large surface-tovolume ratio and highly curved surfaces, the epitaxial stress caused by lattice mismatch can be distributed over a large fraction of the constituent atoms.62 Therefore, the epitaxial growth of CdS on the magic-size CdTe core could successfully passivate surface defects without inducing interface defects, resulting in the high PLQY.

Figure 3. TEM images (a), and absorption and steady-state PL spectra (b) of the CdTe/CdS core/shell QDs with different shell growth times. The emission maximum peak, FWHM, and relative PLQY were plotted as a function of shell growth time (c). Time-resolved PL decay curves (d) of the CdTe/CdS core/shell QDs with different shell growth times. Inset of a: a highresolution TEM image of the CdTe/CdS QDs. The d-spacing of the CdTe/CdS QDs indicates the (220) plane of zinc blende structure of CdS. The excitation wavelengths were 400 nm and 375 nm for the steady-state PL measurement and the time-resolved fluorescence decay measurement, respectively.

Next, we investigated experimental conditions for the DNA functionalization of the CdTe/CdS QDs to increase the number of pt-ssDNA on the surface of QDs while maintaining their optical properties. The number of shell precursors, including Cd-thiolate complexes and pt-ssDNA, was fixed, while CdTe core QDs were diluted from 240 nM to 2.4 nM to vary the molar ratio of


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core-to-shell precursors. After the in situ DNA functionalization during the shell growth, unbound pt-ssDNAs were removed by a centrifugal filter as described in the experimental section. Figure 4 shows the absorption and PL spectra of the synthesized CdTe/CdS QDs with different core concentrations. When the core concentration was decreased from 240 nM to 48 nM, the absorbance at 260 nm was increased, indicating that the number of pt-ssDNA on the QD surface was increased. The calculated numbers of pt-ssDNA on the QD surface were 9.5, 25.1, and 46.6 when the core concentrations were 240, 120, and 48 nM (see details in Figure S4). However, when the concentration of core QDs was 2.4 nM, the synthesized QDs showed a strong absorption maximum peak near 370 nm and a broad PL spectrum, indicating the formation of CdS clusters (Figure S5).63 Consequently, the optimal concentration of core QDs was 120 nM to maximize the pt-ssDNA functionalization while minimizing the formation of CdS clusters.

Figure 4. Absorption (a) and PL spectra (b) of the CdTe/CdS QDs with different core concentration. The absorption spectra were normalized at QDs’ absorption peak of about 585 nm, and the PL spectra were normalized by their peak height. The excitation wavelength was 400 nm for steady-state PL measurements.

The thermal stability of ptDNA-QDs was then examined by measuring the PL spectra of ptDNA-QDs before and after the exposure to the DNA annealing temperature (70 oC) (Figure 5). In this study, the DNA annealing process involves heating at 70 oC for 10 min, followed by incubation at room temperature for 10 min. The heating temperature was set at 70 oC because the KPC target DNA (55 nucleotides) can form a secondary structure which has a melting temperature


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of 42 oC. We also prepared MPA-capped QDs (MPA-QDs) and mtDNA-QDs as control samples. The PL spectra of MPA-QDs showed a red-shifted PL peak with sharply decreased PL intensity after heating at 70oC. These PL features are typically observed when QDs are aggregated. Upon aggregation, the inter-QD distance becomes much shorter, and energy transfer occurs from the smaller QDs to the larger QDs, resulting in red-shifted PL peak and PL quenching.64 Because the colloidal stability of MPA-QDs is maintained by the electrostatic repulsion among the terminal carboxylates of MPA,65 aggregation indicates the detachment of MPA at the elevated temperature. The PL spectra of mtDNA-QDs also showed a decreased PL intensity by 23.9 %, but there was no noticeable PL peak shift presumably because mtDNA was partially dissociated, and the QDs were loosely aggregated. On the other hand, the PL spectra of ptDNA-QDs exhibited no significant changes, indicating that pt-ssDNAs were not detached at the elevated temperature. The changes in the hydrodynamic size of QDs were also monitored after the exposure to the DNA annealing temperature (70 oC) (Figure 6). Note that the initial diameters of DNA-QDs were larger than that of MPA-QDs because of the larger hydrated shell of DNA. No significant change was observed in the hydrodynamic diameter of ptDNA-QDs, indicating excellent thermal stability. However, the hydrodynamic diameters of MPA-QDs and mtDNA-QDs were increased from 2.0 to 10.6 nm and from 10.3 to 26.6 nm, respectively. These results support that aggregation occurred after heating at 70oC Moreover, the smaller rate of changes in the hydrodynamic diameter of mtDNA-QDs compared to that of MPA-QDs can also be explained with loosely aggregated mtDNA-QDs. TEM also reveals that MPA-QDs and mtDNA-QDs aggregate after heating at 70oC (Figure S6).


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Figure 5. Steady-state PL spectra of MPA-QDs (a), mtDNA-QDs (b), and ptDNA-QDs (c) exposed to the DNA annealing temperature (70 oC) for 10 min. The excitation wavelength was 400 nm for steady-state PL measurements.

Figure 6. The hydrodynamic diameter of MPA-QDs (a), mtDNA-QDs (b), and ptDNA-QDs (c) exposed to the DNA annealing temperature (70 oC).

To better understand the higher thermal stability of ptDNA-QD, we compared the binding energies of mt-ssDNA and pt-ssDNA by density functional theory (DFT) calculations. We used monothiolate (CH3(CH2)2S-) and monophosphorothioate ((CH3)2O3PS-) as model structures for mt-ssDNA and pt-ssDNA, respectively (Figure S7). The ligands were placed on a [100] facet of zinc blende CdS to allow interactions with surface cation (Cd2+). Both mtDNA and ptDNA contain


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anionic groups and have strong affinities to cationic surface.66 Three water molecules were explicitly placed near the ligand to reflect the experimental condition, where the solvent effect of water was implicitly applied. As a result, the calculated binding energies of monothiolate and monophosphorothioate are -0.64 eV and -0.25 eV, respectively. Monothiolate has higher binding energy than monophosphorothioate, but pt-ssDNA has seven binding sites.45,46 If we assume that seven phosphorothioate-modified bases of pt-DNA equally contribute to the binding process, the binding energy of pt-ssDNA is roughly estimated by -1.75 eV, which is almost three times larger than that of mt-ssDNA. The results explain how pt-ssDNA remarkably improved the colloidal stability of QDs through the increased binding affinity via multivalent interactions. To illuminate the necessity of thermally stable QDs for DNA detection, we compared the detection of target DNA in the presence and absence of heating at 70 oC during the DNA annealing process. For target DNA detection, the FRET assay solution was prepared by mixing ptDNA-QDs (or mtDNA-QDs as a control sample) with BHQ2-DNA (the FRET acceptor). The target DNA was then mixed with the FRET assay solution, followed by DNA hybridization, as shown in Figure 7. After the hybridization, FRET-induced spectral changes were monitored. The spectral overlap between QDs and BHQ2 are shown in Figure S8. When DNA annealing process involved heating at 70 oC for 10 min prior to incubation at room temperature (70 oC DNA annealing), the FRET efficiency was rapidly increased and saturated in 10 min, whereas annealing of complementary DNA strands at room temperature (RT DNA annealing) resulted in a slowly increased FRET signal (Figure 8a). The result indicates that heating at 70 oC is essentially required to speed up the hybridization and achieve high hybridization efficiency. Note that the FRET efficiency was calculated by 1-ITD+/ITD- with ITD+ and ITD-, which are the PL intensities of QDs with and without target DNA after DNA annealing, respectively. We also conducted the detection of KPC target


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DNA of 55 nucleotides that forms a secondary structure having a melting temperature of 42 oC (Figure 8b). When DNA annealing is performed at RT, the FRET efficiency increased slower compared to the detection of short target DNA, showing that the secondary structure of the DNA hindered the hybridization. Furthermore, detection of the target DNA that forms a more stable secondary structure of a DNA hairpin with 35 basepairs of stem showed no remarkable FRET signals for RT DNA annealing (Figure S9). These results support that heating at 70 oC on hybridization is necessary for faster detection. In addition, mtDNA-QDs showed a similar tendency in DNA annealing time-dependent FRET efficiency, but their FRET efficiencies were lower than that of ptDNA-QDs (Figure 8c & 8d). The lower FRET efficiencies of mtDNA-QDs are ascribed to the PL quenching caused by poor thermal stability as shown in Figure 6.

Figure 7. Working principle of FRET-based DNA detection. When target DNA is added to a mixture of ptDNA-QDs and BHQ2-DNA, sandwich hybridization occurs, resulting in FRETinduced PL quenching.


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Figure 8. DNA annealing time-dependent FRET efficiency during the detection of random sequence target DNA (a) and KPC sequence target DNA (b) with ptDNA-QDs. mtDNA-QDs were also used to detect random sequence target DNA (c) and KPC sequence target DNA (d). The amount of target DNA was 50 nM.

When DNA annealing was conducted, remarkable PL quenching was observed for the mtDNA-QDs with BHQ2-DNA regardless of the presence of target DNA (Figure 9a), which was presumably caused by the non-specific adsorption of BHQ2-DNA on the QD surface. We speculate that the detachment of mtDNA from the QD surface at the annealing temperature (70 oC) might allow the adsorption of BHQ2-DNA. It was reported that O- in the phosphodiester domain can bind to the QD surface,67 supporting the adsorption of BHQ2-DNA. On the other hand, for ptDNAQDs, FRET-induced PL quenching was observed only when target DNAs were added (Figure 9b). No spectral changes were observed without target DNA. To reflect the thermal stability-related PL quenching, we changed the calculation as a “quenching efficiency” which is defined by 1-I/Io with Io and I being the QD PL intensities before and after the DNA annealing, respectively (Figure 9c). The result showed that the poor thermal stability of mtDNA-QDs resulted in the increased


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background noise of 0.4 due to the false-positive signal, while ptDNA-QDs showed the small false-positive signal of 0.05 by the addition of non-complementary ssDNA.

Figure 9. Steady-state PL spectral changes of mtDNA-QDs (a) and ptDNA-QDs (b) mixed with BHQ2-DNA, followed by exposure to the DNA annealing temperature (70 oC) for 10 min. The amount of non-complementary DNA and target DNA was 500 nM. The excitation wavelength was 400 nm for steady-state PL measurements. (c) Calculated quenching efficiency of each FRET assay.

Next, we conducted a FRET assay using Cy5-DNA as a probe DNA, instead of BHQ2DNA, to find more evidence of non-specific adsorption-induced FRET. Because multiple factors other than FRET can cause the PL quenching of QDs, the fluorescence emission of Cy5 by the excitation of QDs can provide direct evidence for FRET. The PL spectra of ptDNA-QDs exhibit no significant spectral changes without target DNA (Figure S10). However, the PL spectra of mtDNA-QDs show the PL quenching of QDs with the increased PL of Cy5 after the exposure to the DNA annealing temperature, 70 oC, for 10 min. Under the same condition, the PL of MPAQDs was dramatically quenched, and that of Cy5 was increased. These results indicate that


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colloidal instability due to the detachment of surface ligands at the DNA annealing temperature caused the non-specific adsorption of Cy5-DNA onto the MPA-QDs and mtDNA-QDs, resulting in false-positive FRET signal. On the other hand, the multivalent phosphorothioate backbone of pt-ssDNA can strongly bind to the QD surface, wrap the QD surface, and prevent the non-specific binding of Cy5-DNA via steric hindrance, resulting in no false FRET signals without target DNA.68 To evaluate the performance of FRET assays, we measured the PL spectra at different target DNA concentrations from 0 to 500 nM. The calibration curve was obtained by plotting the FRET efficiency against the target DNA concentration. When mtDNA-QDs were used, PL quenching was observed after DNA annealing due to the poor thermal stability (Figure 10a), and the background noise was increased to about 0.4, decreasing the FRET signal for the target DNA at low concentrations. The FRET efficiency was increased almost linearly with the target DNA concentration from 8 to 64 nM (Figure 10b). From the linear regression analysis of the calibration curve, the LOD was 16.1 nM, as calculated by 3.29 times the standard deviation of the blank sample measurements (n = 20), which was divided by the slope of the regression line. On the other hand, there were no false signals when ptDNA-QDs were used, and the FRET efficiency was increased almost linearly with increasing the target DNA concentration from 0 to 2 nM (Figure 10c & 10d). As a result, LOD was much lower, 0.47 nM, then that of mtDNA-QDs. In addition, our assay could be applied to detect a well-known multidrug-resistant DNA sequence, KPC, resulting in the LOD of 0.77 nM (Figure S11). Although a conventional fluorescence spectrophotometer was used, the result shows a very high sensitivity comparable to recent works. 13,27-29

We also used a microplate reader, which is advantageous for a high throughput FRET assay,

though it showed some deviations in the PL intensity of the sample depending on the well position. Therefore, the LOD measurement using BHQ2-DNA as a FRET acceptor was not accurate because


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the FRET assay only relies on the PL intensity of QDs. Thus, instead of BHQ2-DNA, we used the Cy5/QD PL intensity ratio as a FRET signal to determine the LOD. The result indicates that the LOD of FRET assay with ptDNA-QDs and, as a pair, Cy5-DNA was 2.15 nM, which is five times higher than that with ptDNA-QDs and BHQ2-DNA (Figure S12).

Figure 10. Steady-state PL spectral changes of mtDNA-QDs (a) and ptDNA-QDs (c) with different amounts of target DNA. Calibration curve and its linear regression in low target DNA concentration regime for mtDNA-QDs (b) and ptDNA-QDs (d).

CONCLUSION We demonstrated that ptDNA-QDs can serve as an efficient donor for the FRET assay for DNA because of their excellent thermal stability at an elevated temperature through multivalent ligand complexation. The multivalent binding of ptDNA to the surface of CdTe/CdS QDs was critically important for surface stabilization and high FRET efficiencies. The DNA-functionalized CdTe/CdS QDs were prepared by in situ DNA functionalization in the aqueous milieu. The synthesized QDs exhibited the high PLQY of above 40 % with the maximum PLQY of 76.9 % at an emission wavelength of 732 nm. The emission of QDs can be tuned from visible to NIR range


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(600 - 750 nm) simply by controlling the shell growth time, allowing us to match their emission spectra with the absorption of a FRET acceptor. The strong binding and steric exclusion of ptssDNA led to higher thermal stability at an elevated temperature and prevented non-specific DNA adsorption, resulting in minimized false positive signal compared to mtDNA-QDs. The target DNA analysis with a conventional fluorescence spectrometer, the LOD of QD-based FRET assay was 0.47 nM. The results suggest that the in situ functionalization of QDs with pt-ssDNA is an effective means to achieve both desirable luminescent properties and colloidal stability, which are required for practical FRET assay for DNA detection.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.XXXX ((please add manuscript number)) • size distribution histogram of QDs; average PL lifetimes of CdTe/CdS QDs; schematic diagram of the band alignment of QD; calculation of the number of DNA per QD; absorption & PL spectra of CdS NCs; TEM of aggregated QDs; 3D model of ligand binding surface of QD; spectral overlap of FRET pairs; thermal stability analysis of QDs with Cy5; FRET assay result for KPC sequence target DNA; FRET assay result with Cy5 acceptor; (PDF))

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (Y.S.N.) and [email protected] (C.A.H.) ORCID Jae Chu Park: 0000-0002-1157-4275 Se Yeon Choi: 0000-0002-4146-6259 Moon Young Yang: 0000-0003-4436-8010 Lin Nan: 0000-0002-4020-7888 Hyebin Na: 0000-0002-6414-2134 Hyun Jung Chung: 0000-0001-5055-902X Cheol Am Hong: 0000-0002-4159-3522 Yoon Sung Nam: 0000-0002-7302-6928 Author Contributions


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Jae Chul Park and Se Yeon Choi contributed equally to this work. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This research was supported by Nano·Material Technology Development Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Science and ICT (NRF2016R1A2B4013045 and NRF-2017M3A7B4052798).

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