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High-conjugation-efficiency aqueous CdSe quantum dots† Cite this: DOI: 10.1039/c3an01198d
Giang H. T. Au,a Wan Y. Shiha and Wei-Heng Shih*b Quantum dots (QDs) are photoluminescent nanoparticles that can be directly or indirectly coupled with a receptor such as an antibody to specifically image a target biomolecule such as an antigen. Recent studies have shown that QDs can be directly made at room temperature and in an aqueous environment (AQDs) with 3-mercaptopropionic acid (MPA) as the capping ligand without solvent and ligand exchange typically required by QDs made by the organic solvent routes (OQDs). In this study, we have synthesized CdSe AQDs and compared their conjugation efficiency and imaging efficacy with commercial carboxylated OQDs in HT29 colon cancer cells using a primary antibody–biotinylated secondary antibody–streptavidin (SA) sandwich. We showed that the best imaging condition for AQDs occurred when one AQD was bound with 3 0.3 SA with a nominal SA/AQD ratio of 4 corresponding to an SA conjugation efficiency of 75 7.5%. In comparison, for commercial CdSe–ZnS OQDs to achieve 2.7 0.4 bound SAs per OQD for comparable imaging efficacy a nominal SA/OQD ratio of 80 was needed corresponding to an SA conjugation efficiency of 3.4 0.5% for CdSe–ZnS OQDs. The more than 10 times better SA conjugation Received 17th June 2013 Accepted 10th September 2013
efficiency of the CdSe AQDs as compared to that of the CdSe–ZnS OQDs was attributed to more capping molecules on the AQD surface as a result of the direct aqueous synthesis. More capping molecules on the AQD surface also allowed the SA–AQD conjugate to be stable in cell culture medium for more than
DOI: 10.1039/c3an01198d
three days without losing their staining capability in a flowing cell culture medium. In contrast, SA–OQD
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conjugates aggregated in cell culture medium and in phosphate buffer saline solution over time.
Introduction Quantum dots (QDs) are semiconducting nanoparticles with unique photoluminescent properties. They are bright and do not photobleach. The wavelength of their emission is tunable with their size through the quantum connement effect. These attributes make them ideal for cellular and molecular imaging applications.1–8 For bioimaging, QDs must be conjugated with a receptor such as an antibody, a peptide, or a nucleotide that binds specically to a target biomolecule such as an antigen,9 a protein,10–13 or a nucleic acid.6 The conjugated QDs must be colloidally stable, and the conjugation of QDs must not interfere with the receptor–target interaction so that conjugated QDs can specically image the target biomolecule. Whether these requirements can be met depends very much on the surface properties of the QDs. Currently, most QDs including the commercial QDs are made in organic solvent,14 which will be
a
School of Biomedical Engineering, Science and Health Systems, Drexel University, 3141 Chestnut Street, Bossone 718, Philadelphia, PA 19104, USA. E-mail: gha23@ drexel.edu; Fax: +1-215-895-4983; Tel: +1-610-362-2563
b
Department of Materials Science and Engineering, Drexel University, 3141 Chestnut Street, Lebow 344, Philadelphia, PA 19104, USA. E-mail: [email protected]; Fax: +1-215-895-6670; Tel: +1-215-895-6636 † Electronic supplementary 10.1039/c3an01198d
information
(ESI)
available.
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termed as OQDs hereaer. OQDs have many attractive attributes such as bright emission with a narrow bandwidth and a uniform particle size. However, the organic-solvent synthesis route is incompatible with the aqueous environment of a biological system. To use OQDs for bioimaging, OQDs must rst undergo ligand exchange and solvent exchange followed by chemically bonding the receptor to a capping molecule on the QD surface in an aqueous environment so that the conjugated QDs can image target biomolecules in a biological system.7,15–19 For example, to synthesize CdSe–ZnS core–shell OQDs, TOPO (trioctylphosphine oxide) is rst purged with owing nitrogen and heated in a reaction ask to 300 C. The stock solution of dimethylcadmium (Me2Cd) and tri-n-octylphosphine selenide (TOPSe) are then added to the ask to form CdSe. Aliquots of the reaction solution are removed at various time intervals to obtain the CdSe nanocrystals of different sizes. CdSe nanocrystals are then precipitated in chloroform and redispersed in hexane. The CdSe nanocrystals in hexane are injected into a ask containing TOPO and trioctylphosphine (TOP) which is then heated to 220 C under owing nitrogen, followed by dropwise addition of diethylzinc (ZnEt2) in TOPO and TOP and hexamethyldisilathiane ((TMS)2S) in TOPO and TOP, stirred and cooled to 90 C for several hours to form the CdSe–ZnS core–shell structure.20 The TOPO-capped CdSe–ZnS OQDs are then precipitated by methanol and redispersed in chloroform. 3-Mercaptopropionic
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Analyst acid (MPA) in chloroform is added to the OQD suspension and made to react for 40 hours. The hydrophilic OQDs are further puried by adding ethyl acetate to the suspension followed by centrifugation and re-dispersion of the pellet in water.21,22 Clearly, with such complex procedures, there is less control of the amount of carboxyl groups on the OQD surface. Consequently, the conjugation of OQDs with receptors is oen accompanied by aggregation23 and requires a high concentration of the receptors.24 Furthermore, capping OQDs with these aqueous-medium compatible ligands by ligand exchange25–27 is not an effective way of populating the capping ligands on the QD surface for efficient conjugation of the receptor to the QDs or the stability of the QDs in an aqueous environment. Recently, Li et al.28–30 pioneered an aqueous synthesis route in which CdS and ZnS QDs were directly synthesized in water with 3-mercaptoproprionic acid (MPA) as the capping molecule (ligand), which will be termed as aqueous QDs (AQDs). AQDs have been shown to be colloidally stable both in vitro28–30 and inside the cells.5,31 It has also been shown that they could be readily conjugated to receptors without the need for ligand or solvent exchange.5,28 This suggests that the aqueous-synthesized AQDs can be advantageous in terms of conjugation and colloidal stability. The main difference between AQDs and OQDs is that AQDs are directly made in an aqueous environment that allows the aqueous-medium compatible capping molecules to directly pack on the AQD surface when the AQDs are being formed, in contrast to ligand exchange processes involved in the OQD system to pack the aqueous-medium compatible capping molecules. In addition, metal ions such as Cd and Zn are known to be chelated with two MPAs under basic conditions.32 Additional MPAs can be packed onto the AQD surface through inclusion of proportional extra metal ions. It is therefore of interest to systematically examine how the aqueous synthesis strategy can affect the conjugation efficiency and colloidal stability of QDs. Again, the terms OQDs and AQDs refer to their initial synthesis routes and not the nal state. The goal of this study is to synthesize MPA-capped CdSe AQDs and compare their protein conjugation efficiency and colloidal stability to those of their OQD counterpart, carboxylated CdSe– ZnS OQDs. We chose MPA-capped CdSe AQDs with a MPA : Cd : Se molar ratio of 4 : 3 : 1 as the model AQDs because the OQD counterpart—carboxylated CdSe–ZnS OQDs—has been extensively studied and widely available commercially. Carboxylated OQDs have been conjugated to a number of biological molecules through chemical or electrostatic linkage.33,34 The QDs can be conjugated to antibodies to directly image the target molecule or conjugated to streptavidin (SA) to indirectly image the target molecule where the SA-linked QDs bind to biotinylated secondary antibodies that are bound to the primary antibodies captured by the target molecules. In this study, we used the indirect imaging protocol involving SA-biotin binding for conjugation efficiency comparison. The model target biomolecule was the Tn-antigen, a truncated glycan carcinoma biomarker of epithelial cancers such as breast,35 gastric,36 ovarian,37 colorectal,38 and pancreatic cancers.39 The model cell line was HT29 colon cancer cells. The nominal SA-to-AQD and SA-to-OQD ratios were varied to identify the optimal ratio for the AQDs and that of
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Paper the OQDs, respectively. Gel electrophoresis was used to quantify the number of SA bound to each QD and to evaluate the conjugation efficiency of the QD. In addition, we measured the zeta potentials and sizes of the AQDs and OQDs before and aer conjugation in phosphate buffer saline (PBS) solution and cell culture media over time to assess their colloidal stability.
Materials and methods 1. Synthesis and characterization of MPA-capped CdSe AQDs A cadmium precursor solution (0.08 M) was rst prepared by dissolving 1.19 g of Cd(NO3)2 powder (Alfa Aesar, Ward Hill, USA) in 50 mL of deionized (DI) water. The selenium precursor solution (0.08 M) was prepared by dissolving 0.315 g of selenium powder (Sigma-Aldrich, MA) and 8.2 g of sodium sulte (Na2SO3) (Sigma-Aldrich, MA) in 50 mL DI water at 80 C under constant stirring for 2 hours until the selenium was completely dissolved. These precursor solutions could be stored for later use. 0.16 mmol of MPA (Sigma-Aldrich, St Louis, MO, USA) was added to 40 mL of DI water and stirred for 10 minutes, followed by adjusting the pH to 11 using tetrapropylammonium hydroxide (TMAH) (Alfar Aesar, Ward Hill, USA). Next, 1 mL of the 0.08 M Cd(NO3)2 precursor was added and stirred for 10 minutes. 1 mL of the selenium precursor was then added to the MPA–Cd mixture, followed by quickly adjusting the pH to 12 and stirring for 10 minutes to precipitate the CdSe AQDs. To further enhance the photoluminescence (PL) intensity, 2 mL of excess 0.08 M Cd(NO3)2 precursor was added to the above CdSe AQD suspensions followed again by a quick adjustment of the pH to 12. The thus-obtained CdSe AQD suspension was clear with a dark yellow tint. The nal concentration of the CdSe AQDs was 1.6 mM in terms of the Se atom with a nominal molar ratio MPA : Cd : Se ¼ 4 : 3 : 1. We have experimented with various MPA : Cd : Se ratios (see ESI†). It was found that CdSe AQDs with MPA : Cd : Se ¼ 4 : 3 : 1 provided the best PL intensity. In what follows, all CdSe AQDs were prepared with a molar ratio of MPA : Cd : Se ¼ 4 : 3 : 1. For storage, the suspension was kept at pH ¼ 12 at 4 C. The PL and the ultraviolet-visible (UV-vis) absorption of the MPA-capped CdSe AQDs were measured using a QM4/2005 spectrouorometer (Photon Technology International, Birmingham, MA, USA) and a USB2000 UV-vis spectrometer (Ocean Optics, NJ, USA) respectively. The X-ray diffraction (XRD) patterns of the AQDs were measured with a Siemens D500 X-ray Diffractometer. The size, morphology and crystalline fringes of the AQDs were examined by transmission electron microscopy (TEM) (JEM-2100, JEOL, West Chester, PA). The quantum yield of the MPA-capped CdSe AQDs was determined by comparing the slope of the total emission intensity versus the total absorption intensity of the AQDs to that of Rhodamine 101 as described below. The size and zeta potential of the SA–AQD conjugates and those of the SA–OQD conjugates were measured using a ZetaSizer (Nano Z, Malvern, Westborough, MA, USA). The AQD particle concentrations were estimated based on an AQD size of 3 nm as evident in the TEM micrograph and a lattice constant of 0.593 nm. Assuming the AQDs to be cubes, an AQD concentration of 1.6 mM based on the Se atom corresponded to an AQD particle concentration of 0.594 mM. This journal is ª The Royal Society of Chemistry 2013
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2. Conjugation of SA to AQDs and OQDs The freshly made AQD suspension was rst stored in a refrigerator (4 C) overnight followed by the removal of free MPAs in the suspension by centrifugation with a 10 kDa lter (Millipore Co., Beillerica, MA) at 3000 rpm for 10 minutes three times. The commercial OQDs, carboxylated CdSe–ZnS (Qdot605-ITK), were purchased from Invitrogen (Grand Island, NY, USA). They are coated with a polymer layer containing –COO surface groups. The polymer layer allows facile dispersion of the OQDs in an aqueous suspension (borate buffer pH ¼ 9). Commercial carboxylated CdSe–ZnS OQDs were used for comparison to the MPA-capped CdSe AQDs. The conjugation procedure of SA to the MPA-capped CdSe AQDs and that to the carboxylated CdSe– ZnS OQDs were similar to that published in the literature40 and is described below. First, N-ethyl-N0 -dimethylaminopropyl-carbodiimide (EDC) (Thermo Scientic, Rockford, IL, USA) and Nhydroxysuccinimide (NHS) (Thermo Scientic, Rockford, IL, USA) were used to facilitate the peptide bond formation between a primary amine of the SA and a carboxyl on the AQD or OQD surface. First, 4 mg of EDC and 6 mg of NHS were dissolved in 1 mL of 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer (TEKNOVA, Hollister, CA) at pH ¼ 6.5. 2 mM of EDC and 5 mM of NHS were added to the suspension of the OQDs (1 mM particle concentration) or that of the AQDs (1.07 mM particle concentration) at pH ¼ 7 in borate buffer. The reaction was incubated for 15 min at room temperature followed by the addition of 2-mercaptoethanol (20 mM) to quench the EDC. The suspension was then run through a desalting column (Zeba Spin 7 KW, Pierce, Rockford, IL) to remove unbound reagents and electrolytes in the suspension. The suspension was then mixed with an SA solution at room temperature and pH ¼ 7.0 for 2 h. To achieve optimal SA/AQD and SA/OQD conjugation, we experimented with SA/AQD molar ratios of 1, 2, 3, 4, 6, and 10, and SA/OQD ratios of 2, 4, 10, 40, 60, and 80. Note for OQDs, the vendor suggested that SA/OQD ratio was 40. For all the molar ratios, the concentrations of AQDs and OQDs were kept at 1 mM. The unused NHS esters bound on the QD surface were then quenched by hydroxylamine hydrochloride (10 mM) (Sigma-Aldrich, St. Louis, MO, USA). Unconjugated QDs and SAs were then removed by microcentrifugation at 12 000 rpm using 100 kDa lters for 5 minutes ve times. Aer each microcentrifugation, the volume of the suspension was restored with a 50 mM borate buffer solution of pH 8.3. Aer ve consecutive microcentrifugations, the suspension was ltered through a syringe with a 0.2 mm lter (Fisher Scientic, Newark, DE) to remove large aggregates if any. The conjugated AQD and OQD suspensions were then stored at 4 C before use. 3. Custom polyacrylamine/agarose gel system To characterize the SA–AQD and SA–OQD conjugates, a custom gel system consisting of a revolving gel with 2% polyacrylamide and 0.5% agarose and a stacking gel with 1% polyacrylamide and 0.5% agarose was made following the procedures of Liu et al.23 All the reagents for the custom gel system were purchased from Fisher BioReagents (Allentown, PA, USA). The
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Analyst revolving gel was made by mixing 1% agarose solution in distilled water with an equal volume of 4% of 37.5/1 molar ratio acrylamide/bis acrylamide solution in a 2 tris/boric acid/EDTA (TBE) buffer solution at 60 C and by adding 0.05% (by weight) ammonium persulfate and 0.04% (by volume) of tetramethylethylenediamine (TEMED) right before pouring. The stacking gel which consisted of 1% polyacrylamide with 0.5% agarose was prepared in a similar fashion as the revolving gel and was poured aer the revolving gel had solidied completely (0.5– 1 hour). Aer purication, 15 mL each SA–QD complex was loaded into the wells of the gel at 1 mM in terms of QD concentration. 4. Immunouorescent (IF) staining of xed HT29 cells HT29 cells (ATCC, Manassas, VA, USA) which expressed Tn antigen were grown on cover glasses overnight and then xed with 4% paraformaldehyde for 15 minutes followed by washing with Tris buffer saline containing 0.1% Tween (TBS). A biotin/ streptavidin kit (Vector Laboratories, Burlingame, CA) was used to block endogenous biotin in cells. Cells were then washed with TBS three times and blocked with 10% normal goat serum (Jackson ImmunoResearch, West Grove, PA) for 1 hour at room temperature. Mouse anti-Tn antigen antibody (1 : 20 dilution, Genetex, CA) was added and incubated for 1 hour at room temperature to bind to the Tn antigen on the cell surface followed by washing with TBS three times to remove unbound anti-Tn antibodies. Biotinylated goat anti-mouse antibody (1 : 50 dilution, Invitrogen, Grand Island, NY) was then added to the cells for 30 minutes at room temperature to bond to the mouse anti-Tn antibody. Unbound biotinylated goat anti-mouse antibody was then removed by washing with TBS three times. SA–AQD (or SA–OQD) conjugates were then added to the cells for 30 minutes to bind to the biotinylated goat anti-mouse antibody. Finally, cells were washed with TBS three times to remove the unbound SA–AQDs (or SA–OQDs) and countermounted with mounting medium containing DAPI (Vector Laboratories, Burlingame, CA).
5. Stability of SA–AQD and SA–OQD complexes For biological application of the functionalized AQDs, the conjugated AQD complexes must be stable under biological conditions. To study the stability of SA-conjugated AQDs (SA– AQD) and SA-conjugated OQDs, we used SA–AQD complexes with SA/AQD ¼ 4 and the SA–OQD complexes with SA/OQD ¼ 80 as model complexes because at these ratios AQDs and OQDs exhibited comparable uorescent intensity per cell in imaging Tn-antigen on HT29 cells as is shown below. The SA–AQD and SA–OQD complexes were added into the cell culture media with 10% fetal bovine serum (FBS) and incubated at 37 C for 24 h, 48 h, and 72 h. It has been known that OQDs form large aggregates in cell media due to the binding of OQDs with the proteins.41 In this study, the size of the SA–AQD complexes and that of the SA–OQD complexes as a function of time as well as the zeta potential of the SA–AQD complexes and that of the SA– OQD complexes were examined using ZetaSizer (Malvern, MA).
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Results and discussion
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1. MPA-capped CdSe AQDs The PL and UV-vis absorption spectra of the MPA-capped CdSe AQDs and carboxylated CdSe–ZnS OQDs are shown in Fig. 1a. As can be seen, the emission of the AQDs peaked at 610 nm (which was close to the 605 nm emission peak of the OQDs) when excited at 460 nm. The luminescence of a MPA-capped AQD suspension on a UV lamp appeared yellow-orange color as shown in the inset of Fig. 1a. This is due to the broad bandwidth of the emission peak compared to the narrow emission spectrum of OQDs, which resulted in red uorescent color. The broad bandwidth of CdSe AQDs is predominantly due to defectstate emission42 while the narrow emission of OQDs is due to band edge emission. The yellow-orange color of AQDs on a UV lamp is due to the broad emission bandwidth. If a cut-off lter of 600 nm was applied, the AQDs would appear red instead of yellow-orange. In Fig. 1b, we showed the integrated PL intensity versus the absorption at 460 nm of the AQDs and OQDs together with that of Rhodamine 101 (Rd 101) as a reference. Rd 101 QY is more or less independent of the excitation wavelength in the range of 460–565 nm.43–45 We used the measurement of Rhodamine 101 at 565 nm as the standard. Using this standard, the QY of Rhodamine 101 when excited at 460 nm was 96 2.1%. The quantum yield (QY) of the AQDs was determined using the following eqn (1) (ref. 46) fQD
! hQD 2 GradQD ¼ fRd GradRd hRd 2
(1)
GradQD and GradRd denote the slope of the integrated emission intensity (i.e., the total area under the emission peak instead of the peak height) of the AQDs and that of the Rd 101, versus absorbance of the AQDs and that of the Rd 101 in Fig. 1b, respectively, and fQD and fRd are the refractive index of the medium of the AQD suspension which was water (hence hQD ¼ 1.33)46 and that of the solvent of the Rd 101 solution, which was ethanol (hence, hRd ¼ 1.36),46 respectively. The QY of the AQDs as determined from Fig. 1b was 70 2.5% with the QY of Rd 101 being 96 2.1%,47 comparable to that of the commercial CdSe–ZnS OQDs which was 81.6 2.5%. The reason that the current AQDs had such a high QY was associated with the optimal MPA : Cd : Se molecular ratio in the present study. Note that even though Fig. 1a shows that the emission peak height of the OQDs was about six times that of the AQDs, the emission peak width of the AQDs was about three times that of the OQDs. Aer integrating the area under the emission peak, the integrated PL intensity of the AQDs was roughly half that of the OQDs at the same concentration, which was similar to the ratio of the absorbance between the AQDs and OQDs at 460 nm (see the absorption spectra in Fig. 1a). As a result, the quantum yield of the AQDs and that of the OQDs are similar. The crystalline structure of the AQDs was the zincblende structure as evidenced by the X-ray diffraction (XRD) pattern shown in Fig. 1c. From the size distribution and TEM micrograph of the AQDs shown in Fig. 1d, one can see that the AQDs had a size of about 3 nm. Note that if the QDs were to
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exhibit edge-state emissions, 3 nm QDs would emit green light of 540 nm (ref. 48 and 49) rather than yellow/orange light of broad bandwidth around 600 nm as shown in Fig. 1a. This is because the present AQDs exhibited defect-state emission involving energy difference from the bottom of the conduction band to the defect states inside the band gap, which is smaller than the band gap (the energy difference involved in edge-state emission).
2. SA–AQD and SA–OQD conjugation We examined the conjugation of SA to AQDs and that of SA to OQDs under the conditions where there were more SAs than AQDs or OQDs. To characterize the SA–AQD and SA–OQD conjugates, aer the nal ltration with a 100 kD lter, we ran the retentate and the ltrate of each SA–AQD and SA–OQD conjugate through the custom-made gel. Fig. 2a shows the uorescence of the QDs in the SA–AQD and SA–OQD conjugates from the retentate taken with FluorChem E (Protein Simple, San Jose, CA). Coomassie blue (Brilliant R250, Across Organic, NJ, USA) was used to visualize the SA. Fig. 2b shows the image of SA in the SA–AQD and SA–OQD conjugates from the retentate. A protein ladder was included in lane 15 of the bright eld image in Fig. 2b. Lane 1 in Fig. 2b contained free SA which appeared as a dark band at about 55 kDa in the bright eld image but not in the uorescent image of lane 1 in Fig. 2a. Lane 2 contained free AQDs, which were present in the uorescent image in Fig. 2a but not in the bright eld image in Fig. 2b since no protein was present. Lane 3 to 7 contained SA–AQD conjugates with SA–AQD number ratios 3, 4, 6 and 10 respectively. Lane 8–13 contained SA–OQD conjugates with SA–OQD number ratios 2, 4, 10, 40, 60 and 80 respectively. As can be seen from the bright eld images in Fig. 2b, both SA–AQD conjugates with SA/AQD ¼ 3, 4, and 6 and SA–OQD conjugates with SA/OQD ¼ 40, 60, and 80 exhibited a thick band in the range of 130–250 kD, which was much larger than the SA (55 kD) and QD (80% maximal SA conjugation efficiency at nominal SA/AQD ¼ 4 while the maximal SA conjugation efficiency was only 6% at nominal SA/OQD ¼ 80. *Optimal SA/QD ratios.
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SA–OQD conjugates with an SA/OQD ¼ 40, 60 and 80 obtained in a BX51 Olympus uorescent microscope. Using images similar to those shown in Fig. 5a, the uorescent intensity per cell was further analyzed using ImageJ and averaged over 100 cells for each SA–AQD and SA–OQD conjugates. In Fig. 5b, we plot the average uorescent intensity per cell versus the nominal SA–AQD for AQDs and that versus the nominal SA/OQD ratio for OQDs. As can be seen, the uorescent intensity per cell could reach 320 with SA–AQD conjugates with nominal SA/AQD ¼ 4 in which there were 3 0.3 bound SAs per AQD (see Fig. 3a). In contrast, the uorescent intensity per cell could only reach 350 by SA–OQD conjugates with a nominal SA/OQD ¼ 80. Clearly, high protein conjugation efficiency was an advantage of the AQDs over OQDs as AQDs needed only one twentieth of the SA needed by OQDs to achieve a similar staining result. Finally, although the optimal number of conjugated SA per AQD was similar to that of conjugated SA per OQD, the resultant staining signal was slightly higher with the OQDs possibly due to the fact that the OQDs had a higher QY than the AQDs. We hypothesize that the easier conjugation of SA to AQDs than to OQDs was a result of more carboxyl groups on the AQDs than on the OQDs due to the in situ incorporation of the MPAs as the capping molecules in the AQD synthesis process. In contrast, carboxyl groups on the OQDs needed to be incorporated aer synthesis by ligand exchange. To compare the amount of the bound carboxyl groups on the AQDs to that on the OQDs, we carried out Fourier transform infrared (FTIR) spectroscopy on AQD and OQD suspensions of the same concentration. Both the AQD and OQD suspensions were ltered to remove unbound MPA if any. The resultant FTIR spectra of AQDs (black line) and that of OQDs (red line) are shown in Fig. 6. Since all free capping molecules and ions were removed and the water background was subtracted, the peaks in the remaining spectra could only be attributed to the QDs. The experiment was performed with the same volume and concentration for both OQDs and AQDs. The carboxylic acid O–H stretch appeared as a very broad band in the region of 3300–2500 cm1. The carbonyl stretch C]O appeared from 1760–1690 cm1 (ref. 50) in both AQDs and OQDs with AQDs exhibiting stronger peaks. As can be seen, the AQDs exhibit a much stronger O–H bend and C–O stretch peaks at around 1440–1395 cm1 and 1320–1210 cm1 regions and a C]O peak at around 1750 cm1.50 The O–H band and C–O stretch peaks were the results of MPA which was four times stronger than the carboxylated surface on OQDs when the intensities of these peaks were integrated, indicating that indeed AQDs had more carboxylated groups on the surface. The reason AQDs had four times more carboxylated groups on the surface was that the AQDs were deliberately made with three times more Cd than Se. In addition, each of the two excess Cds per Se was paired with two excess MPAs as can be seen from the nominal molar ratio, MPA : Cd : Se of 4 : 3 : 1. Under basic conditions each excess Cd was chelated with two MPAs.32 The MPA-chelated excess Cd would mostly sit on top of the Se on the AQD surface, forming a MPA-chelated Cd “shell” on the AQD surface. The fact that the CdSe AQDs had a size of 3 nm size and that the nominal molar ratio, MPA : Cd : Se of 4 : 3 : 1 support the notion that there was
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Fig. 5 Immunofluorescent staining of HT29 colon cancer cells by SA–QD conjugates. (a) HT29 cells stained by SA–QD conjugates to image Tn antigen on the cell surface with various nominal SA/AQD and SA/OQD ratios. Conjugates with nominal SA–AQD ¼ 4 gave the brightest images; (b) relative fluorescent intensity per cell versus nominal SA–AQD and nominal SA–OQD. * Optimal SA/QD ratio.
a MPA-chelated Cd “shell” on the CdSe AQDs. In addition, given that the emission of the AQDs was due to surface trap states, the notion that the AQDs had a MPA-chelated Cd “shell” was also consistent with the fact that the emission of the AQDs increased with an increasing amount of extra Cd and MPA (with a 1 : 2 Cd : MPA molar ratio).28 Note that packing an extra amount of MPA on the AQD surface through the chelation of extra MPA to extra Cd precursor was only possible in an aqueous environment. Furthermore, we also measured the zeta potential of the AQDs and that of the OQDs in PBS at pH 7. The zeta potential of the AQDs in PBS was found to be 30 mV and that of the OQDs to be 22 mV. AQDs were more negatively charged than OQDs which was consistent with the above FTIR results and further
Fig. 6 FTIR spectrum of AQDs (black) and that of OQDs (red) with a focus on the capping carboxyl group. The carboxylic acid O–H stretch appears as a very broad band in the region of 3300–2500 cm1. The carbonyl stretch C]O appears at 1760–1690 cm1. The C–O stretch in the region of 1320–1210 cm1 and the O–H bend in the region of 1440–1395 cm1 indicate the presence of the surface carboxyl group on the AQD and OQD surfaces. Integrated peak intensities indicated that ADQs had 4 times more COOH groups on the surface compared to OQDs.
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supported the hypothesis that more MPA were on the AQDs than on the OQDs. 4. Stability of SA–AQD and SA–OQD conjugates In order to use SA–AQD and SA–OQD conjugates as molecular probes to image living systems, they must be stable under physiological conditions.17 There have been very few studies addressing this aspect. We measured the size of the SA–AQD conjugates with SA/AQD ¼ 4 and that of the SA–OQD conjugates with a SA/OQD ¼ 80 in a cell culture medium with 10% FBS at 37 C and in PBS every 24 h over a 72 h period for comparison. The results are shown in Fig. 7. As can be seen the size of the SA– AQD conjugates remained at around 32 nm in both the cell culture medium and in PBS throughout the 72 h period indicating that there was no observable aggregation of the SA–AQD conjugates in the cell medium (full squares) or in the PBS (open squares). In contrast, the size of the SA–OQD conjugates increased with time in both PBS and in cell medium indicating that the SA–OQD conjugates were less stable. By 72 hours, the size of the SA–OQD was increased to about 80 nm and 160 nm in PBS and in cell medium, respectively, which is consistent with the literature that OQDs were shown to bind to albumin nonspecically in cell media resulting in large aggregates and reduced PL intensity. We also examined the binding capability of cell culture medium-aged SA-conjugated AQDs to stain live cells in a ow that mimicked physiological conditions. Live HT29 colon cancer cells grown on a cover glass were pre-treated with the mouse anti-Tn antibody and biotinylated goat anti-mouse antibody and placed in a ow chamber at 37 in an incubator (MCO-20AIC, Sanyo, Japan). A schematic of the ow system is shown in Fig. 8. Note that all cell slides were blocked with 1% bovine serum albumin (BSA), and a biotin/streptavidin kit before exposure to the treatment of antibodies and SA–AQD conjugates as described in the Experimental section. The SA– AQD conjugates of SA/AQD ¼ 4 were aged in a cell culture medium at room temperature for 24, 48, and 72 h before they
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Fig. 7 Stability of SA–AQD and SA–OQD conjugates in PBS and in cell media for up to 72 hours after conjugation. SA–AQD conjugates were stable in both PBS and cell media with little change in size whereas SA–OQD conjugates were only stable for 24 h and started to show signs of aggregation after 48 h in PBS and 24 h in cell media.
were introduced into the sample holder of the ow system to stain HT29 cells in the ow chamber in a ow driven by a peristaltic pump (7712062, Cole-Parmer, Chicago, IL) at a ow rate of 1 mL min1 for 30 min. Figs. 9a–c show the uorescent micrographs of the HT29 cells stained by the SA–AQD conjugates aged in a cell culture medium for 24, 48, and 72 h, respectively. A negative control is shown in Fig. 9d where HT29 cells were untreated with mouse anti-Tn AQD conjugates. Fig. 9e and f show the micrographs of HT29 cells stained without ow at room temperature by SA–AQD conjugates aged in PBS and in cell culture medium for 2 h, respectively. Note that the blue color in these gures was due to the DAPI staining of the nuclei and the red color was due to the uorescence of the AQDs. As can be seen, all the micrographs in Fig. 9a–f were similar, indicating that the SA–AQD conjugates were stable in cell media with serum for the whole 72 h period and the SA– AQD conjugates were able to specically stain the Tn-antigen on the cell surface under such ow conditions, another piece of evidence for the stability of the SA–AQD complex in the cell culture media.
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Fig. 9 Immunofluorescent staining of the Tn antigen on the HT29 colon cancer cell surface in a flow system. SA–AQD conjugates were incubated in cell-culture for (a) 24 hours, (b) 48 hours, and (c) 72 hours before circulation and exposure to cells. (d) Negative control: no primary antibody to allow for specific binding. Positive controls without circulation: (e) SA–AQD conjugates were incubated in PBS for 24 hours prior to exposure to the cells. (f) SA–AQD conjugates were incubated in cell culture for 24 hours prior to exposure to cells. Blue: DAPI for nucleus, red: SA–AQD for Tn-antigen expression. Scale bar ¼ 80 mm.
Conclusions We have examined the conjugation and imaging efficacy of MPA-capped CdSe AQDs and compared them to those of CdSe– ZnS OQDs. The optimal nominal SA/AQD ratio of 4 yielded one AQD bound with 3 0.3 SAs with 75 7.5% SA conjugation efficiency. The optimal nominal SA/OQD ratio of 80 yielded one OQD bound with 2.7 0.4 SAs with 3.4 0.5% SA conjugation. The ease of conjugation of the AQDs – i.e., 10 fold fewer SAs were needed for AQDs than for OQDs – was attributed to the fact that AQDs had more MPA capping molecules on the surface than OQDs, a result of the direct aqueous synthesis of the AQDs, as evidenced in the FTIR study as well as zeta potential measurements. The SA–AQD conjugates were shown to be stable in PBS as well as in cell media at room temperature for 72 h in contrast with the SA–OQD conjugates whose size grew with time in both PBS and cell culture media. We have also shown that the SA–AQD conjugates were also stable with no diminishing imaging efficacy in owing cell culture media at 37 C, pH ¼ 7 for 72 h.
Acknowledgements This work is supported in part by the Grants W81XWH-09-10701 of Department of Defense and the Drexel University-Wallace Coulter Foundation Partnership. The authors would like to thank Mr Wei Wu for his help in setting up the ow system, Mr Song Han for his help in obtaining the XRD pattern, and Dr Greg Johnson for his help in calculating the particle lattice constant. Fig. 8 A schematic of the flow system consisting of a peristaltic pump, a SA– AQD conjugate sample holder, and a flow chamber containing live HT29 cells on a cover-slit. SA–AQD conjugates were added in the sample holder and circulated in the flow driven by a peristaltic pump to reach the cells in the flow chamber.
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Notes and references 1 J. K. Jaiswal, E. R. Goldman, H. Mattoussi and S. M. Simon, Nat. Methods, 2004, 1, 73–78.
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High-conjugation-efficiency aqueous CdSe quantum dots† Cite this: DOI: 10.1039/c3an01198d
Giang H. T. Au,a Wan Y. Shiha and Wei-Heng Shih*b Quantum dots (QDs) are photoluminescent nanoparticles that can be directly or indirectly coupled with a receptor such as an antibody to specifically image a target biomolecule such as an antigen. Recent studies have shown that QDs can be directly made at room temperature and in an aqueous environment (AQDs) with 3-mercaptopropionic acid (MPA) as the capping ligand without solvent and ligand exchange typically required by QDs made by the organic solvent routes (OQDs). In this study, we have synthesized CdSe AQDs and compared their conjugation efficiency and imaging efficacy with commercial carboxylated OQDs in HT29 colon cancer cells using a primary antibody–biotinylated secondary antibody–streptavidin (SA) sandwich. We showed that the best imaging condition for AQDs occurred when one AQD was bound with 3 0.3 SA with a nominal SA/AQD ratio of 4 corresponding to an SA conjugation efficiency of 75 7.5%. In comparison, for commercial CdSe–ZnS OQDs to achieve 2.7 0.4 bound SAs per OQD for comparable imaging efficacy a nominal SA/OQD ratio of 80 was needed corresponding to an SA conjugation efficiency of 3.4 0.5% for CdSe–ZnS OQDs. The more than 10 times better SA conjugation Received 17th June 2013 Accepted 10th September 2013
efficiency of the CdSe AQDs as compared to that of the CdSe–ZnS OQDs was attributed to more capping molecules on the AQD surface as a result of the direct aqueous synthesis. More capping molecules on the AQD surface also allowed the SA–AQD conjugate to be stable in cell culture medium for more than
DOI: 10.1039/c3an01198d
three days without losing their staining capability in a flowing cell culture medium. In contrast, SA–OQD
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conjugates aggregated in cell culture medium and in phosphate buffer saline solution over time.
Introduction Quantum dots (QDs) are semiconducting nanoparticles with unique photoluminescent properties. They are bright and do not photobleach. The wavelength of their emission is tunable with their size through the quantum connement effect. These attributes make them ideal for cellular and molecular imaging applications.1–8 For bioimaging, QDs must be conjugated with a receptor such as an antibody, a peptide, or a nucleotide that binds specically to a target biomolecule such as an antigen,9 a protein,10–13 or a nucleic acid.6 The conjugated QDs must be colloidally stable, and the conjugation of QDs must not interfere with the receptor–target interaction so that conjugated QDs can specically image the target biomolecule. Whether these requirements can be met depends very much on the surface properties of the QDs. Currently, most QDs including the commercial QDs are made in organic solvent,14 which will be
a
School of Biomedical Engineering, Science and Health Systems, Drexel University, 3141 Chestnut Street, Bossone 718, Philadelphia, PA 19104, USA. E-mail: gha23@ drexel.edu; Fax: +1-215-895-4983; Tel: +1-610-362-2563
b
Department of Materials Science and Engineering, Drexel University, 3141 Chestnut Street, Lebow 344, Philadelphia, PA 19104, USA. E-mail: [email protected]; Fax: +1-215-895-6670; Tel: +1-215-895-6636 † Electronic supplementary 10.1039/c3an01198d
information
(ESI)
available.
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See
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termed as OQDs hereaer. OQDs have many attractive attributes such as bright emission with a narrow bandwidth and a uniform particle size. However, the organic-solvent synthesis route is incompatible with the aqueous environment of a biological system. To use OQDs for bioimaging, OQDs must rst undergo ligand exchange and solvent exchange followed by chemically bonding the receptor to a capping molecule on the QD surface in an aqueous environment so that the conjugated QDs can image target biomolecules in a biological system.7,15–19 For example, to synthesize CdSe–ZnS core–shell OQDs, TOPO (trioctylphosphine oxide) is rst purged with owing nitrogen and heated in a reaction ask to 300 C. The stock solution of dimethylcadmium (Me2Cd) and tri-n-octylphosphine selenide (TOPSe) are then added to the ask to form CdSe. Aliquots of the reaction solution are removed at various time intervals to obtain the CdSe nanocrystals of different sizes. CdSe nanocrystals are then precipitated in chloroform and redispersed in hexane. The CdSe nanocrystals in hexane are injected into a ask containing TOPO and trioctylphosphine (TOP) which is then heated to 220 C under owing nitrogen, followed by dropwise addition of diethylzinc (ZnEt2) in TOPO and TOP and hexamethyldisilathiane ((TMS)2S) in TOPO and TOP, stirred and cooled to 90 C for several hours to form the CdSe–ZnS core–shell structure.20 The TOPO-capped CdSe–ZnS OQDs are then precipitated by methanol and redispersed in chloroform. 3-Mercaptopropionic
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Analyst acid (MPA) in chloroform is added to the OQD suspension and made to react for 40 hours. The hydrophilic OQDs are further puried by adding ethyl acetate to the suspension followed by centrifugation and re-dispersion of the pellet in water.21,22 Clearly, with such complex procedures, there is less control of the amount of carboxyl groups on the OQD surface. Consequently, the conjugation of OQDs with receptors is oen accompanied by aggregation23 and requires a high concentration of the receptors.24 Furthermore, capping OQDs with these aqueous-medium compatible ligands by ligand exchange25–27 is not an effective way of populating the capping ligands on the QD surface for efficient conjugation of the receptor to the QDs or the stability of the QDs in an aqueous environment. Recently, Li et al.28–30 pioneered an aqueous synthesis route in which CdS and ZnS QDs were directly synthesized in water with 3-mercaptoproprionic acid (MPA) as the capping molecule (ligand), which will be termed as aqueous QDs (AQDs). AQDs have been shown to be colloidally stable both in vitro28–30 and inside the cells.5,31 It has also been shown that they could be readily conjugated to receptors without the need for ligand or solvent exchange.5,28 This suggests that the aqueous-synthesized AQDs can be advantageous in terms of conjugation and colloidal stability. The main difference between AQDs and OQDs is that AQDs are directly made in an aqueous environment that allows the aqueous-medium compatible capping molecules to directly pack on the AQD surface when the AQDs are being formed, in contrast to ligand exchange processes involved in the OQD system to pack the aqueous-medium compatible capping molecules. In addition, metal ions such as Cd and Zn are known to be chelated with two MPAs under basic conditions.32 Additional MPAs can be packed onto the AQD surface through inclusion of proportional extra metal ions. It is therefore of interest to systematically examine how the aqueous synthesis strategy can affect the conjugation efficiency and colloidal stability of QDs. Again, the terms OQDs and AQDs refer to their initial synthesis routes and not the nal state. The goal of this study is to synthesize MPA-capped CdSe AQDs and compare their protein conjugation efficiency and colloidal stability to those of their OQD counterpart, carboxylated CdSe– ZnS OQDs. We chose MPA-capped CdSe AQDs with a MPA : Cd : Se molar ratio of 4 : 3 : 1 as the model AQDs because the OQD counterpart—carboxylated CdSe–ZnS OQDs—has been extensively studied and widely available commercially. Carboxylated OQDs have been conjugated to a number of biological molecules through chemical or electrostatic linkage.33,34 The QDs can be conjugated to antibodies to directly image the target molecule or conjugated to streptavidin (SA) to indirectly image the target molecule where the SA-linked QDs bind to biotinylated secondary antibodies that are bound to the primary antibodies captured by the target molecules. In this study, we used the indirect imaging protocol involving SA-biotin binding for conjugation efficiency comparison. The model target biomolecule was the Tn-antigen, a truncated glycan carcinoma biomarker of epithelial cancers such as breast,35 gastric,36 ovarian,37 colorectal,38 and pancreatic cancers.39 The model cell line was HT29 colon cancer cells. The nominal SA-to-AQD and SA-to-OQD ratios were varied to identify the optimal ratio for the AQDs and that of
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Paper the OQDs, respectively. Gel electrophoresis was used to quantify the number of SA bound to each QD and to evaluate the conjugation efficiency of the QD. In addition, we measured the zeta potentials and sizes of the AQDs and OQDs before and aer conjugation in phosphate buffer saline (PBS) solution and cell culture media over time to assess their colloidal stability.
Materials and methods 1. Synthesis and characterization of MPA-capped CdSe AQDs A cadmium precursor solution (0.08 M) was rst prepared by dissolving 1.19 g of Cd(NO3)2 powder (Alfa Aesar, Ward Hill, USA) in 50 mL of deionized (DI) water. The selenium precursor solution (0.08 M) was prepared by dissolving 0.315 g of selenium powder (Sigma-Aldrich, MA) and 8.2 g of sodium sulte (Na2SO3) (Sigma-Aldrich, MA) in 50 mL DI water at 80 C under constant stirring for 2 hours until the selenium was completely dissolved. These precursor solutions could be stored for later use. 0.16 mmol of MPA (Sigma-Aldrich, St Louis, MO, USA) was added to 40 mL of DI water and stirred for 10 minutes, followed by adjusting the pH to 11 using tetrapropylammonium hydroxide (TMAH) (Alfar Aesar, Ward Hill, USA). Next, 1 mL of the 0.08 M Cd(NO3)2 precursor was added and stirred for 10 minutes. 1 mL of the selenium precursor was then added to the MPA–Cd mixture, followed by quickly adjusting the pH to 12 and stirring for 10 minutes to precipitate the CdSe AQDs. To further enhance the photoluminescence (PL) intensity, 2 mL of excess 0.08 M Cd(NO3)2 precursor was added to the above CdSe AQD suspensions followed again by a quick adjustment of the pH to 12. The thus-obtained CdSe AQD suspension was clear with a dark yellow tint. The nal concentration of the CdSe AQDs was 1.6 mM in terms of the Se atom with a nominal molar ratio MPA : Cd : Se ¼ 4 : 3 : 1. We have experimented with various MPA : Cd : Se ratios (see ESI†). It was found that CdSe AQDs with MPA : Cd : Se ¼ 4 : 3 : 1 provided the best PL intensity. In what follows, all CdSe AQDs were prepared with a molar ratio of MPA : Cd : Se ¼ 4 : 3 : 1. For storage, the suspension was kept at pH ¼ 12 at 4 C. The PL and the ultraviolet-visible (UV-vis) absorption of the MPA-capped CdSe AQDs were measured using a QM4/2005 spectrouorometer (Photon Technology International, Birmingham, MA, USA) and a USB2000 UV-vis spectrometer (Ocean Optics, NJ, USA) respectively. The X-ray diffraction (XRD) patterns of the AQDs were measured with a Siemens D500 X-ray Diffractometer. The size, morphology and crystalline fringes of the AQDs were examined by transmission electron microscopy (TEM) (JEM-2100, JEOL, West Chester, PA). The quantum yield of the MPA-capped CdSe AQDs was determined by comparing the slope of the total emission intensity versus the total absorption intensity of the AQDs to that of Rhodamine 101 as described below. The size and zeta potential of the SA–AQD conjugates and those of the SA–OQD conjugates were measured using a ZetaSizer (Nano Z, Malvern, Westborough, MA, USA). The AQD particle concentrations were estimated based on an AQD size of 3 nm as evident in the TEM micrograph and a lattice constant of 0.593 nm. Assuming the AQDs to be cubes, an AQD concentration of 1.6 mM based on the Se atom corresponded to an AQD particle concentration of 0.594 mM. This journal is ª The Royal Society of Chemistry 2013
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2. Conjugation of SA to AQDs and OQDs The freshly made AQD suspension was rst stored in a refrigerator (4 C) overnight followed by the removal of free MPAs in the suspension by centrifugation with a 10 kDa lter (Millipore Co., Beillerica, MA) at 3000 rpm for 10 minutes three times. The commercial OQDs, carboxylated CdSe–ZnS (Qdot605-ITK), were purchased from Invitrogen (Grand Island, NY, USA). They are coated with a polymer layer containing –COO surface groups. The polymer layer allows facile dispersion of the OQDs in an aqueous suspension (borate buffer pH ¼ 9). Commercial carboxylated CdSe–ZnS OQDs were used for comparison to the MPA-capped CdSe AQDs. The conjugation procedure of SA to the MPA-capped CdSe AQDs and that to the carboxylated CdSe– ZnS OQDs were similar to that published in the literature40 and is described below. First, N-ethyl-N0 -dimethylaminopropyl-carbodiimide (EDC) (Thermo Scientic, Rockford, IL, USA) and Nhydroxysuccinimide (NHS) (Thermo Scientic, Rockford, IL, USA) were used to facilitate the peptide bond formation between a primary amine of the SA and a carboxyl on the AQD or OQD surface. First, 4 mg of EDC and 6 mg of NHS were dissolved in 1 mL of 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer (TEKNOVA, Hollister, CA) at pH ¼ 6.5. 2 mM of EDC and 5 mM of NHS were added to the suspension of the OQDs (1 mM particle concentration) or that of the AQDs (1.07 mM particle concentration) at pH ¼ 7 in borate buffer. The reaction was incubated for 15 min at room temperature followed by the addition of 2-mercaptoethanol (20 mM) to quench the EDC. The suspension was then run through a desalting column (Zeba Spin 7 KW, Pierce, Rockford, IL) to remove unbound reagents and electrolytes in the suspension. The suspension was then mixed with an SA solution at room temperature and pH ¼ 7.0 for 2 h. To achieve optimal SA/AQD and SA/OQD conjugation, we experimented with SA/AQD molar ratios of 1, 2, 3, 4, 6, and 10, and SA/OQD ratios of 2, 4, 10, 40, 60, and 80. Note for OQDs, the vendor suggested that SA/OQD ratio was 40. For all the molar ratios, the concentrations of AQDs and OQDs were kept at 1 mM. The unused NHS esters bound on the QD surface were then quenched by hydroxylamine hydrochloride (10 mM) (Sigma-Aldrich, St. Louis, MO, USA). Unconjugated QDs and SAs were then removed by microcentrifugation at 12 000 rpm using 100 kDa lters for 5 minutes ve times. Aer each microcentrifugation, the volume of the suspension was restored with a 50 mM borate buffer solution of pH 8.3. Aer ve consecutive microcentrifugations, the suspension was ltered through a syringe with a 0.2 mm lter (Fisher Scientic, Newark, DE) to remove large aggregates if any. The conjugated AQD and OQD suspensions were then stored at 4 C before use. 3. Custom polyacrylamine/agarose gel system To characterize the SA–AQD and SA–OQD conjugates, a custom gel system consisting of a revolving gel with 2% polyacrylamide and 0.5% agarose and a stacking gel with 1% polyacrylamide and 0.5% agarose was made following the procedures of Liu et al.23 All the reagents for the custom gel system were purchased from Fisher BioReagents (Allentown, PA, USA). The
This journal is ª The Royal Society of Chemistry 2013
Analyst revolving gel was made by mixing 1% agarose solution in distilled water with an equal volume of 4% of 37.5/1 molar ratio acrylamide/bis acrylamide solution in a 2 tris/boric acid/EDTA (TBE) buffer solution at 60 C and by adding 0.05% (by weight) ammonium persulfate and 0.04% (by volume) of tetramethylethylenediamine (TEMED) right before pouring. The stacking gel which consisted of 1% polyacrylamide with 0.5% agarose was prepared in a similar fashion as the revolving gel and was poured aer the revolving gel had solidied completely (0.5– 1 hour). Aer purication, 15 mL each SA–QD complex was loaded into the wells of the gel at 1 mM in terms of QD concentration. 4. Immunouorescent (IF) staining of xed HT29 cells HT29 cells (ATCC, Manassas, VA, USA) which expressed Tn antigen were grown on cover glasses overnight and then xed with 4% paraformaldehyde for 15 minutes followed by washing with Tris buffer saline containing 0.1% Tween (TBS). A biotin/ streptavidin kit (Vector Laboratories, Burlingame, CA) was used to block endogenous biotin in cells. Cells were then washed with TBS three times and blocked with 10% normal goat serum (Jackson ImmunoResearch, West Grove, PA) for 1 hour at room temperature. Mouse anti-Tn antigen antibody (1 : 20 dilution, Genetex, CA) was added and incubated for 1 hour at room temperature to bind to the Tn antigen on the cell surface followed by washing with TBS three times to remove unbound anti-Tn antibodies. Biotinylated goat anti-mouse antibody (1 : 50 dilution, Invitrogen, Grand Island, NY) was then added to the cells for 30 minutes at room temperature to bond to the mouse anti-Tn antibody. Unbound biotinylated goat anti-mouse antibody was then removed by washing with TBS three times. SA–AQD (or SA–OQD) conjugates were then added to the cells for 30 minutes to bind to the biotinylated goat anti-mouse antibody. Finally, cells were washed with TBS three times to remove the unbound SA–AQDs (or SA–OQDs) and countermounted with mounting medium containing DAPI (Vector Laboratories, Burlingame, CA).
5. Stability of SA–AQD and SA–OQD complexes For biological application of the functionalized AQDs, the conjugated AQD complexes must be stable under biological conditions. To study the stability of SA-conjugated AQDs (SA– AQD) and SA-conjugated OQDs, we used SA–AQD complexes with SA/AQD ¼ 4 and the SA–OQD complexes with SA/OQD ¼ 80 as model complexes because at these ratios AQDs and OQDs exhibited comparable uorescent intensity per cell in imaging Tn-antigen on HT29 cells as is shown below. The SA–AQD and SA–OQD complexes were added into the cell culture media with 10% fetal bovine serum (FBS) and incubated at 37 C for 24 h, 48 h, and 72 h. It has been known that OQDs form large aggregates in cell media due to the binding of OQDs with the proteins.41 In this study, the size of the SA–AQD complexes and that of the SA–OQD complexes as a function of time as well as the zeta potential of the SA–AQD complexes and that of the SA– OQD complexes were examined using ZetaSizer (Malvern, MA).
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Results and discussion
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1. MPA-capped CdSe AQDs The PL and UV-vis absorption spectra of the MPA-capped CdSe AQDs and carboxylated CdSe–ZnS OQDs are shown in Fig. 1a. As can be seen, the emission of the AQDs peaked at 610 nm (which was close to the 605 nm emission peak of the OQDs) when excited at 460 nm. The luminescence of a MPA-capped AQD suspension on a UV lamp appeared yellow-orange color as shown in the inset of Fig. 1a. This is due to the broad bandwidth of the emission peak compared to the narrow emission spectrum of OQDs, which resulted in red uorescent color. The broad bandwidth of CdSe AQDs is predominantly due to defectstate emission42 while the narrow emission of OQDs is due to band edge emission. The yellow-orange color of AQDs on a UV lamp is due to the broad emission bandwidth. If a cut-off lter of 600 nm was applied, the AQDs would appear red instead of yellow-orange. In Fig. 1b, we showed the integrated PL intensity versus the absorption at 460 nm of the AQDs and OQDs together with that of Rhodamine 101 (Rd 101) as a reference. Rd 101 QY is more or less independent of the excitation wavelength in the range of 460–565 nm.43–45 We used the measurement of Rhodamine 101 at 565 nm as the standard. Using this standard, the QY of Rhodamine 101 when excited at 460 nm was 96 2.1%. The quantum yield (QY) of the AQDs was determined using the following eqn (1) (ref. 46) fQD
! hQD 2 GradQD ¼ fRd GradRd hRd 2
(1)
GradQD and GradRd denote the slope of the integrated emission intensity (i.e., the total area under the emission peak instead of the peak height) of the AQDs and that of the Rd 101, versus absorbance of the AQDs and that of the Rd 101 in Fig. 1b, respectively, and fQD and fRd are the refractive index of the medium of the AQD suspension which was water (hence hQD ¼ 1.33)46 and that of the solvent of the Rd 101 solution, which was ethanol (hence, hRd ¼ 1.36),46 respectively. The QY of the AQDs as determined from Fig. 1b was 70 2.5% with the QY of Rd 101 being 96 2.1%,47 comparable to that of the commercial CdSe–ZnS OQDs which was 81.6 2.5%. The reason that the current AQDs had such a high QY was associated with the optimal MPA : Cd : Se molecular ratio in the present study. Note that even though Fig. 1a shows that the emission peak height of the OQDs was about six times that of the AQDs, the emission peak width of the AQDs was about three times that of the OQDs. Aer integrating the area under the emission peak, the integrated PL intensity of the AQDs was roughly half that of the OQDs at the same concentration, which was similar to the ratio of the absorbance between the AQDs and OQDs at 460 nm (see the absorption spectra in Fig. 1a). As a result, the quantum yield of the AQDs and that of the OQDs are similar. The crystalline structure of the AQDs was the zincblende structure as evidenced by the X-ray diffraction (XRD) pattern shown in Fig. 1c. From the size distribution and TEM micrograph of the AQDs shown in Fig. 1d, one can see that the AQDs had a size of about 3 nm. Note that if the QDs were to
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exhibit edge-state emissions, 3 nm QDs would emit green light of 540 nm (ref. 48 and 49) rather than yellow/orange light of broad bandwidth around 600 nm as shown in Fig. 1a. This is because the present AQDs exhibited defect-state emission involving energy difference from the bottom of the conduction band to the defect states inside the band gap, which is smaller than the band gap (the energy difference involved in edge-state emission).
2. SA–AQD and SA–OQD conjugation We examined the conjugation of SA to AQDs and that of SA to OQDs under the conditions where there were more SAs than AQDs or OQDs. To characterize the SA–AQD and SA–OQD conjugates, aer the nal ltration with a 100 kD lter, we ran the retentate and the ltrate of each SA–AQD and SA–OQD conjugate through the custom-made gel. Fig. 2a shows the uorescence of the QDs in the SA–AQD and SA–OQD conjugates from the retentate taken with FluorChem E (Protein Simple, San Jose, CA). Coomassie blue (Brilliant R250, Across Organic, NJ, USA) was used to visualize the SA. Fig. 2b shows the image of SA in the SA–AQD and SA–OQD conjugates from the retentate. A protein ladder was included in lane 15 of the bright eld image in Fig. 2b. Lane 1 in Fig. 2b contained free SA which appeared as a dark band at about 55 kDa in the bright eld image but not in the uorescent image of lane 1 in Fig. 2a. Lane 2 contained free AQDs, which were present in the uorescent image in Fig. 2a but not in the bright eld image in Fig. 2b since no protein was present. Lane 3 to 7 contained SA–AQD conjugates with SA–AQD number ratios 3, 4, 6 and 10 respectively. Lane 8–13 contained SA–OQD conjugates with SA–OQD number ratios 2, 4, 10, 40, 60 and 80 respectively. As can be seen from the bright eld images in Fig. 2b, both SA–AQD conjugates with SA/AQD ¼ 3, 4, and 6 and SA–OQD conjugates with SA/OQD ¼ 40, 60, and 80 exhibited a thick band in the range of 130–250 kD, which was much larger than the SA (55 kD) and QD (80% maximal SA conjugation efficiency at nominal SA/AQD ¼ 4 while the maximal SA conjugation efficiency was only 6% at nominal SA/OQD ¼ 80. *Optimal SA/QD ratios.
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SA–OQD conjugates with an SA/OQD ¼ 40, 60 and 80 obtained in a BX51 Olympus uorescent microscope. Using images similar to those shown in Fig. 5a, the uorescent intensity per cell was further analyzed using ImageJ and averaged over 100 cells for each SA–AQD and SA–OQD conjugates. In Fig. 5b, we plot the average uorescent intensity per cell versus the nominal SA–AQD for AQDs and that versus the nominal SA/OQD ratio for OQDs. As can be seen, the uorescent intensity per cell could reach 320 with SA–AQD conjugates with nominal SA/AQD ¼ 4 in which there were 3 0.3 bound SAs per AQD (see Fig. 3a). In contrast, the uorescent intensity per cell could only reach 350 by SA–OQD conjugates with a nominal SA/OQD ¼ 80. Clearly, high protein conjugation efficiency was an advantage of the AQDs over OQDs as AQDs needed only one twentieth of the SA needed by OQDs to achieve a similar staining result. Finally, although the optimal number of conjugated SA per AQD was similar to that of conjugated SA per OQD, the resultant staining signal was slightly higher with the OQDs possibly due to the fact that the OQDs had a higher QY than the AQDs. We hypothesize that the easier conjugation of SA to AQDs than to OQDs was a result of more carboxyl groups on the AQDs than on the OQDs due to the in situ incorporation of the MPAs as the capping molecules in the AQD synthesis process. In contrast, carboxyl groups on the OQDs needed to be incorporated aer synthesis by ligand exchange. To compare the amount of the bound carboxyl groups on the AQDs to that on the OQDs, we carried out Fourier transform infrared (FTIR) spectroscopy on AQD and OQD suspensions of the same concentration. Both the AQD and OQD suspensions were ltered to remove unbound MPA if any. The resultant FTIR spectra of AQDs (black line) and that of OQDs (red line) are shown in Fig. 6. Since all free capping molecules and ions were removed and the water background was subtracted, the peaks in the remaining spectra could only be attributed to the QDs. The experiment was performed with the same volume and concentration for both OQDs and AQDs. The carboxylic acid O–H stretch appeared as a very broad band in the region of 3300–2500 cm1. The carbonyl stretch C]O appeared from 1760–1690 cm1 (ref. 50) in both AQDs and OQDs with AQDs exhibiting stronger peaks. As can be seen, the AQDs exhibit a much stronger O–H bend and C–O stretch peaks at around 1440–1395 cm1 and 1320–1210 cm1 regions and a C]O peak at around 1750 cm1.50 The O–H band and C–O stretch peaks were the results of MPA which was four times stronger than the carboxylated surface on OQDs when the intensities of these peaks were integrated, indicating that indeed AQDs had more carboxylated groups on the surface. The reason AQDs had four times more carboxylated groups on the surface was that the AQDs were deliberately made with three times more Cd than Se. In addition, each of the two excess Cds per Se was paired with two excess MPAs as can be seen from the nominal molar ratio, MPA : Cd : Se of 4 : 3 : 1. Under basic conditions each excess Cd was chelated with two MPAs.32 The MPA-chelated excess Cd would mostly sit on top of the Se on the AQD surface, forming a MPA-chelated Cd “shell” on the AQD surface. The fact that the CdSe AQDs had a size of 3 nm size and that the nominal molar ratio, MPA : Cd : Se of 4 : 3 : 1 support the notion that there was
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Fig. 5 Immunofluorescent staining of HT29 colon cancer cells by SA–QD conjugates. (a) HT29 cells stained by SA–QD conjugates to image Tn antigen on the cell surface with various nominal SA/AQD and SA/OQD ratios. Conjugates with nominal SA–AQD ¼ 4 gave the brightest images; (b) relative fluorescent intensity per cell versus nominal SA–AQD and nominal SA–OQD. * Optimal SA/QD ratio.
a MPA-chelated Cd “shell” on the CdSe AQDs. In addition, given that the emission of the AQDs was due to surface trap states, the notion that the AQDs had a MPA-chelated Cd “shell” was also consistent with the fact that the emission of the AQDs increased with an increasing amount of extra Cd and MPA (with a 1 : 2 Cd : MPA molar ratio).28 Note that packing an extra amount of MPA on the AQD surface through the chelation of extra MPA to extra Cd precursor was only possible in an aqueous environment. Furthermore, we also measured the zeta potential of the AQDs and that of the OQDs in PBS at pH 7. The zeta potential of the AQDs in PBS was found to be 30 mV and that of the OQDs to be 22 mV. AQDs were more negatively charged than OQDs which was consistent with the above FTIR results and further
Fig. 6 FTIR spectrum of AQDs (black) and that of OQDs (red) with a focus on the capping carboxyl group. The carboxylic acid O–H stretch appears as a very broad band in the region of 3300–2500 cm1. The carbonyl stretch C]O appears at 1760–1690 cm1. The C–O stretch in the region of 1320–1210 cm1 and the O–H bend in the region of 1440–1395 cm1 indicate the presence of the surface carboxyl group on the AQD and OQD surfaces. Integrated peak intensities indicated that ADQs had 4 times more COOH groups on the surface compared to OQDs.
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supported the hypothesis that more MPA were on the AQDs than on the OQDs. 4. Stability of SA–AQD and SA–OQD conjugates In order to use SA–AQD and SA–OQD conjugates as molecular probes to image living systems, they must be stable under physiological conditions.17 There have been very few studies addressing this aspect. We measured the size of the SA–AQD conjugates with SA/AQD ¼ 4 and that of the SA–OQD conjugates with a SA/OQD ¼ 80 in a cell culture medium with 10% FBS at 37 C and in PBS every 24 h over a 72 h period for comparison. The results are shown in Fig. 7. As can be seen the size of the SA– AQD conjugates remained at around 32 nm in both the cell culture medium and in PBS throughout the 72 h period indicating that there was no observable aggregation of the SA–AQD conjugates in the cell medium (full squares) or in the PBS (open squares). In contrast, the size of the SA–OQD conjugates increased with time in both PBS and in cell medium indicating that the SA–OQD conjugates were less stable. By 72 hours, the size of the SA–OQD was increased to about 80 nm and 160 nm in PBS and in cell medium, respectively, which is consistent with the literature that OQDs were shown to bind to albumin nonspecically in cell media resulting in large aggregates and reduced PL intensity. We also examined the binding capability of cell culture medium-aged SA-conjugated AQDs to stain live cells in a ow that mimicked physiological conditions. Live HT29 colon cancer cells grown on a cover glass were pre-treated with the mouse anti-Tn antibody and biotinylated goat anti-mouse antibody and placed in a ow chamber at 37 in an incubator (MCO-20AIC, Sanyo, Japan). A schematic of the ow system is shown in Fig. 8. Note that all cell slides were blocked with 1% bovine serum albumin (BSA), and a biotin/streptavidin kit before exposure to the treatment of antibodies and SA–AQD conjugates as described in the Experimental section. The SA– AQD conjugates of SA/AQD ¼ 4 were aged in a cell culture medium at room temperature for 24, 48, and 72 h before they
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Fig. 7 Stability of SA–AQD and SA–OQD conjugates in PBS and in cell media for up to 72 hours after conjugation. SA–AQD conjugates were stable in both PBS and cell media with little change in size whereas SA–OQD conjugates were only stable for 24 h and started to show signs of aggregation after 48 h in PBS and 24 h in cell media.
were introduced into the sample holder of the ow system to stain HT29 cells in the ow chamber in a ow driven by a peristaltic pump (7712062, Cole-Parmer, Chicago, IL) at a ow rate of 1 mL min1 for 30 min. Figs. 9a–c show the uorescent micrographs of the HT29 cells stained by the SA–AQD conjugates aged in a cell culture medium for 24, 48, and 72 h, respectively. A negative control is shown in Fig. 9d where HT29 cells were untreated with mouse anti-Tn AQD conjugates. Fig. 9e and f show the micrographs of HT29 cells stained without ow at room temperature by SA–AQD conjugates aged in PBS and in cell culture medium for 2 h, respectively. Note that the blue color in these gures was due to the DAPI staining of the nuclei and the red color was due to the uorescence of the AQDs. As can be seen, all the micrographs in Fig. 9a–f were similar, indicating that the SA–AQD conjugates were stable in cell media with serum for the whole 72 h period and the SA– AQD conjugates were able to specically stain the Tn-antigen on the cell surface under such ow conditions, another piece of evidence for the stability of the SA–AQD complex in the cell culture media.
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Fig. 9 Immunofluorescent staining of the Tn antigen on the HT29 colon cancer cell surface in a flow system. SA–AQD conjugates were incubated in cell-culture for (a) 24 hours, (b) 48 hours, and (c) 72 hours before circulation and exposure to cells. (d) Negative control: no primary antibody to allow for specific binding. Positive controls without circulation: (e) SA–AQD conjugates were incubated in PBS for 24 hours prior to exposure to the cells. (f) SA–AQD conjugates were incubated in cell culture for 24 hours prior to exposure to cells. Blue: DAPI for nucleus, red: SA–AQD for Tn-antigen expression. Scale bar ¼ 80 mm.
Conclusions We have examined the conjugation and imaging efficacy of MPA-capped CdSe AQDs and compared them to those of CdSe– ZnS OQDs. The optimal nominal SA/AQD ratio of 4 yielded one AQD bound with 3 0.3 SAs with 75 7.5% SA conjugation efficiency. The optimal nominal SA/OQD ratio of 80 yielded one OQD bound with 2.7 0.4 SAs with 3.4 0.5% SA conjugation. The ease of conjugation of the AQDs – i.e., 10 fold fewer SAs were needed for AQDs than for OQDs – was attributed to the fact that AQDs had more MPA capping molecules on the surface than OQDs, a result of the direct aqueous synthesis of the AQDs, as evidenced in the FTIR study as well as zeta potential measurements. The SA–AQD conjugates were shown to be stable in PBS as well as in cell media at room temperature for 72 h in contrast with the SA–OQD conjugates whose size grew with time in both PBS and cell culture media. We have also shown that the SA–AQD conjugates were also stable with no diminishing imaging efficacy in owing cell culture media at 37 C, pH ¼ 7 for 72 h.
Acknowledgements This work is supported in part by the Grants W81XWH-09-10701 of Department of Defense and the Drexel University-Wallace Coulter Foundation Partnership. The authors would like to thank Mr Wei Wu for his help in setting up the ow system, Mr Song Han for his help in obtaining the XRD pattern, and Dr Greg Johnson for his help in calculating the particle lattice constant. Fig. 8 A schematic of the flow system consisting of a peristaltic pump, a SA– AQD conjugate sample holder, and a flow chamber containing live HT29 cells on a cover-slit. SA–AQD conjugates were added in the sample holder and circulated in the flow driven by a peristaltic pump to reach the cells in the flow chamber.
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