Imaging Quantum Dots using FUJIFILM LAS 4000 Application Note John Pizzonia, Ph.D. 9-28-07
Quantum dots (also known as nanocrystals) are a special class of materials known as semiconductors, which are crystals composed of periodic group II-VI, III-V, or IV-VI metals. They are small molecular entities, ranging from 2-20 nanometers (10-50 atoms) in diameter, putting them between small dye molecules and fluorescent proteins (Figure One). Quantum dots are available as Evifluors (Evident Technologies and Millipore Corp) and While they are available from a wide variety of sources the two primary suppliers of both native and biocongugated forms are Invitrogen (i.e. Qdots ) and Evident Technologies (Evifluors). Quantum Dots 2-20nm Figure One: Size perspective of Quantum Dots compared to other Biological entities. Quantum dots are very bright and particularly well suited to function as labels in Life Science applications due to their high quantum yeild. The dots confine electrons that can be excited by light, as these electrons return to lower energy levels they fluoresce. Quantum dots have extremely broad excitation spectra and narrow emission spectra. This unique characteristic of quantum dots, compared to traditional organic fluorochromes (i.e. fluorescein, rhodamine and cyanines), results in a very large Stokes shift (Figure Two) and the Excitation and Emission Spectral Profiles Quantum dots Organic Fluorochromes Figure Two: Comparison of Stoke s shifts in quantum dots versus traditional organic fluorochromes. 2
ability to be excited by a single wavelength light source. The excitation profiles of indivdual Qdots and Evifluors are summarized in Figure Three. Wavelength (nm) Invitrogen Wavelength (nm) Evident Figure Three: Excitation spectra for Qdots (left) and Evifluors (right). For most quantum dots UV (365nm) or blue light (460nm) is all that is necessary for maximal excitation. This is because quantum dot output or tunability is almost exclusively a function of size (see Figure Four). The larger the inorganic core of the quantum dot the longer the wavelength of emission upon excitation from a single UV source. Figure Four: Tunability of quantum dot output as a function of size The inorganic core is typically composed of metals such as cadmium, selenium and gadillium (Figure Five (A)). This core is usually capped with an inoganic shell composed of materials like zinc sulfide. 3
A B Figure Five: Anatomy of a quantum dot (A). Electron micrograph of individual quantum dots (B). The inorganic core is next made water soluble for use in life science applications by coating the quantum dot surface with PEG lipids. This also prevents the quantum dots from self aggregation (Figure Five (B)). Finally, the lipid coated cores can be functionalized with a carboxyl or amine linker group for subsequent conjugation to other bio molecules. There are now many groups that have been developing targeted applications using quantum dots and the field remains one of the most promising areas for optical imaging in the future! Quantum dots linked to biological molecules, such as antibodies, have shown promise as a new tools for detecting and quantifying a wide variety of molecules in both invitro (i.e. Western blotting) and invivo (i.e. cancer biomarker detection) applications. It won t be long before the use of so-called bioconjugated quantum dots may finally be ready for widespread use in the clinic. The remainder of this discussion will focus on the use of quantum dots in invitro applications such as Western Blotting. The LAS4000 is a CCD-based chemiluminescent and fluorescent imager than is ideally suited for detection of quantum dots in both invitro and invivo applications. This versitile system has both UV (365nm) and Blue (460nm) LED panels (Figure Five) to provide epiillumination for excitation of blots and/or gels labeled with quantum dots. 4
Figure Five: Spectral output for epi illumination LED panels available in the LAS4000. The spectral graphs shown in Figure Two suggest that while the UV line produces maximal exciation for most of the shorter wavelength quantum dots a broad range of excitation wavelengths can be used for excitation, particularly for the longer wavelength quantum dots. Experiments with the LAS4000 underscore the value of UV but also demonstrate that the the larger 800nm Qdot is very effectively excited by the Blue 460nm LED panels (Figure Six). Excitation of Qdot 565nm Measure Net Intensity (AU) 400000 350000 300000 250000 200000 150000 100000 50000 0 UV-365nm 460nm 530nm Excitation Qdot 800nm Measured Net Intensity (AU) 250000 200000 150000 100000 50000 0 460nm UV-365nm 635nm 535nm 720nm Figure 6: Comparison of excitation wavelengths for 565nm (top) and 800nm (bottom) quantum dots. 5
For this experiment Qdots were diluted 1:1000 from stock and were next slot-blotted in a serial 1:2 dilution onto Immobilon-FL membranes. Membranes were imaged on the Fuji LAS4000 using the indicated light source for 0.25secs. The emitted signal was filtered with 565WB20 (top) and 820AF50 (bottom) bandpass filters respectively. Using these excitation options there are a number of potential quantum dots that can be used alone or multiplexed in combinations to produce 2 or more reporter signals in a Western blotting application. Table One outlines the bioconjugated quantum dots offered as Evifluors and Qdots. Table One: List of Commerically Available Bioconjugated Quantum dots. Evifluors Name Adirondack Green Fort Orange Maple Red-Orange Jonamac Red Emission 520nm 600nm 620nm 680nm Species Goat anti-mouse, Goat anti-rabbit, Goat anti-rat, Also available conjugated to Biotin or Streptavidin. Qdots Emission 525nm 565nm 585nm 605nm 655nm 705nm 800nm Species Goat anti-mouse, Goat anti-rabbit, Goat anti-rat, Goat anti-human. Also available conjugated to Biotin and Prepared in other host species. This information can be used in conjunction with the spectral profiles for the filters currently available for the LAS4000 (Figure Eight) to help design multiplexing stratgies for Western blotting protocols. Bandpass Emission Filters Other Emission Filters Y515 670LP Figure Eight: Spectral properties of filters currently available for the LAS4000. 6
Once you have selected your labels the next decision to make is the choice of membrane. Traditional nitrocellulose provides low background and resultant potential signal to noise (S/N), but is fragile and can warp causing aberrations in downstream imaging steps. Millipore Corporation released a Polyvinylidene Difluoride (PVDF) which had excellent structural properties, and which works very well for chemiluminescent protocols, but which produced much higher backgrounds than nitrocellulose in fluorescence based applications. Millipore subsequently released an improved formulation of the PVDF (Immobilon-FL) that has backgrounds at or below that of traditional nitrocellulose. Images of individual slot blots captured on the LAS4000 confirmed this observation. As shown in Figure Nine 565nm Qdots conjugated to species specific antibodies, slotblotted onto the indicated membrane, produced comparable levels of detection sensitivity S/N. In this particular experiment the both membranes were excited using 360nm Epi-UV panels. Emitted signal was filtered with the 565WB20 bandpass filter. Based on these results Immobilon-FL is routinely used in the lab and was used in all subsequent experiments reported. Immobilon-FL Relative Dilution 1:100 1:1000 1:100 1:1000 1:2 Nitrocellulose Figure Nine: 565nm qdots were slot-blotted onto the indicated membrane. Membranes were imaged on the Fuji LAS4000. Excitation using 360nm Epi-UV panels. Emitted signal was filtered with a 565WB20 bandpass filter. Quantum dots not only have the improved analytical accuracy that fluorescence reporters share in general like increased linear dynamic range for the expanded assay accuracy, but they are also more photostable (e.g. reduced self quenching and no photobleaching) as compared to traditional organic fluorchromes. The fluorescence lifetime is orders of magnitude longer than typical autofluoresence lifetimes and many multiples of typical organic dye lifetimes. Still the question of whether quantum dots possession the same ultimate detection sensitivity compared to the more traditional chemiluminescent approach is usually always the first one asked. Experiments performed in our laboratories 7
and summarized in Figure Nine demonstrate that quantum dots have comparable detection capability for equivalent amounts of Western blotted protein while delivering much better analytical accuracy (dynamic range). Qdot 800 Chemi Qdot 800 12000000 1:2 Measured Net Intens 10000000 8000000 6000000 4000000 2000000 R 2 = 0.9996 0 0 20 40 60 80 100 120 Re la tive dilution * * Measured Net Intensity Alkaline Phosphatase 3.00E+08 2.50E+08 2.00E+08 1.50E+08 1.00E+08 R 2 = 0.6655 5.00E+07 0.00E+00 0 20 40 60 80 100 120 Re la tive Dilution Figure Nine: Sensitivity comparison between Invitrogen s 800nm Qdot and Alkaline Phosphatase chemiluminescence. Normal mouse serum was diluted 1:100,000 and serially tritated in steps of 1:2. Membranes were blocked with a non-mammilian based reagent and hybridized with anti-mouse secondary antibodies and 1:5,000. Blots were exposed for 10min with no binning. With good detection sensitivity demonstrated by next obvious advantage that can be designed into Western blotting protocols using quantum dots is multiplexing. To evaluate this 3 quantum dots with non-overlapping emission spectra were identified and appropriate bandpass filters were designed. The filters produced by Omega Optical were 565WB20, 646AF20, and 820AF50 to be used in conjunction with the 565nm, 655nm, and 800nm Qdots respectively (Figure Ten). Figure Ten: Spectral profiles for 565WB20, 646AF20, and 820AF50 filters produced by Omega Optical overlayed on emission spectra for 565nm, 655nm, and 800nm Qdots. 8
The relative detection sensitivity of the LAS4000 system for several quantum dots was evaluated by making serial dilutions from a 1:1000 dilution titrated 1:2. 565nm 655nm 800nm 1:2 Figure Eleven: 565nm 655nm and 800nm Qdots slot-blotted onto Immobilon-FL membranes were imaged on the Fuji LAS4000. Excitation = Blue 460nm epi-panels,. Exposure time =.25secs. Emitted signal was filtered with 565WB20 645AF20 and 820AF50 bandpass filters respectively. Results (Figure Eleven) show roughly equivalent detection sensitivities indicating that these quantum dots can be used reliably for designing multiplexed Western blotting based experiments. To demonstrate the workflow mouse and rabbit immunoglobulin (2.5, 0.83, and 0.16 µg) were separated via SDSPAGE and Western blotted onto Immoblin-FL membrane. Membranes were probed with anti-mouse and anti-rabbit antibodies preconjugated to 800nm and 565nm Qdots. Results (Figure Twelve) show good sensitivity and selectivity can be achieved using simple Western blotting protocols. IgG species Mouse Rabbit µg/lane 2.5 0.83 0.16 2.5 0.83 0.16 Figure Twelve: Western blotted IgG detected with 800nm anti-mouse and 565nm anti-rabbit Qdots. 9
An increasing number of research reports are underscoring the value that quantum dot nanocrystals bring to the researchers tool box. The information reported in this Application Note demonstrate that conventional Western blotting techniques can be easily optimized using quantum dots from a variety of reliable sources as lables to produce uniquly detectable signals. These signals can all be cost effectively detected using the LAS4000 fluorescence imager analyzer. For more information please vist http://fujifilmlifescienceusa.com/. 10