Article pubs.acs.org/Langmuir
Fluorescence Detection and Imaging of Biomolecules Using the Micropatterned Nanostructured Aluminum Oxide Xiang Li, Yuan He, and Long Que* Institute for Micromanufacturing, Louisiana Tech University, Ruston, Louisiana 71272, United States S Supporting Information *
ABSTRACT: Micropatterns of the nanostructured aluminum oxide (NAO) with sizes from 5 to 200 μm have been successfully fabricated on the indium tin oxide (ITO) glass substrate by simply combining a lift-off process and a one-step anodization process for the first time. The detection of fluorescent dyes and biomolecules tagged with fluorescent dyes on the NAO has been investigated and demonstrated successfully. Experiments reveal that the micropatterned NAO substrates can increase the fluorescence signals up to 2 or 3 orders of magnitude compared to the glass substrate, suggesting a possibility to significantly reduce the consumption of the biosamples for fluorescence-based sensing, imaging, and analysis. The stability of the NAO substrates for fluorescence enhancement has also been evaluated by monitoring the fluorescence signals after the fluorophores applied on the substrates for a period of time and reusing the same NAO substrates many times. It was found that this type of substrate has very good stability. Because the micropatterned NAO can be easily integrated with microsensors or microfluidic chips, a simple and inexpensive fluorescence enhancement platform can be developed for a variety of applications, such as microarray technology and single-cell imaging, facilitating the construction of the on-chip fluorescencebased micro- or nanosystems.
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INTRODUCTION Even though the labeling of fluorescent dyes to biomolecules is a relatively complicated work, given its high sensitivity and multiplexing capability, fluorescence sensing and detection have been one of the most widely used technologies in many different fields, such as medical imaging (e.g., cancer cell imaging) and biological detection and sensing (e.g., DNA arrays and gene sequencing), just to name a few.1,2 While the optimization of the performance of the fluorophores or the use of quantum dots is one approach to further enhance the sensitivity and stability of the fluorescence technology, the exploration of the advanced nanomaterial- and nanostructurebased substrates as enhancers of the fluorescence signals is another viable approach to achieve the same goal. Even though both approaches are important and useful for the detection of biomolecules of ultralow concentration, the latter approach would be particularly useful to facilitate the on-chip fluorescence detection and imaging if the advanced fluorescence substrate can be readily integrated with microdevices. Among the advanced fluorescence substrates, the metallic (Au, Ag, and Al) nanostructure substrates3−6 and some semiconducting or metallic oxide nanostructure substrates7−9 show great promise. For instance, Au, Ag, or Al nanoparticles or nanostructures have been widely used as fluorescence enhancers. The physical mechanism for the metal-enhanced fluorescence (MEF) is due to the interactions of the excited fluorophores with surface plasmon resonances in metals. However, the fluorophores have to be located in close proximity to but cannot be directly on the surface of the © 2013 American Chemical Society
metallic nanostructures to avoid the quenching effect. Hence, a thin layer of dielectric material (e.g., SiO2 of 5 nm) usually has to be used as a spacer between the metallic nanostructures and the fluorophores. Examples of MEF substrates, such as silver fractal nanostructures,3 gold surface nanoscale grating,4 aluminum bowtie nanoapertures,5 and gold plasmonic nanoantenna dots,6 have been successfully demonstrated by several groups. However, some of the advanced fluorescence substrates have the following shortcomings. One is the expensive material, such as Au or Ag, used for constructing the substrates. The other is that the expensive nanofabrication process, such as electron-beam lithography or focused ion-beam milling, is required to fabricate the nanostructure substrates, thereby preventing the production of inexpensive nanostructure substrates for fluorescence enhancement in a cost-effective manner. On the other hand, some non-metallic nanomaterials and nanostructures, which have also been discovered and exploited to increase the fluorescence signals and sensitivity, may offer alternative cost-effective technical platforms in that no expensive nanofabrication process is necessarily required. Some representative examples, such as the nanoscaled zinc oxide (ZnO) substrate,7 ZnO/SiO2 core/shell nanorod substrate,8 and nanoscaled SnO2 substrate,9 have been demonstrated for fluorescence enhancement successfully. Since anodic aluminum oxide (AAO) was discovered about 2 Received: December 6, 2012 Revised: January 21, 2013 Published: January 22, 2013 2439
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Figure 1. (a−c) Representative optical micrographs and SEM images of the fabricated NAO micropatterns with different shapes and sizes. (d-1) SEM images of a circular NAO micropattern. (d-2 and d-3) SEM images of the close-up of the NAO, showing nanoscale aluminum oxide grains with nanopores distributed among them.
decades ago,10 it has been widely used for a variety of applications because of its unique nanostructures and optical properties. One application is for label-free biosensing based on the optical interference, resulting from its ordered nanohole arrays.11−13 Another interesting application is to develop a nanoscale matrix luminescence converter of ionizing radiation to visible light by embedding the terbium- and strontium-doped titania xerogel in the AAO honeycombs.14,15 In addition, fluorescence enhancement by inserting the fluorescent dyes or the fluorescent dye-labeled biomolecules inside the nanopores has also been achieved using a Kretschmann−Raether (ATR) setup.16,17 Recently, the fluorescence enhancement has been observed by directly depositing the fluorescent dyes on the AAO surface by our group.18 Herein, the fabrication of micropatterns of the nanostructured aluminum oxide (NAO) on glass substrate is reported, and the fluorescence detection and imaging of fluorescent dyes and labeled biomolecules using the NAO micropatterns are demonstrated for the first time. Experimental results reveal that the NAO substrates may provide a new avenue for achieving highly sensitive fluorescence detection. In addition, given its simple and inexpensive fabrication process and its simplicity and compatibility to be integrated with microdevices and systems, this technology may open up a great deal of applications for high-throughput, highly sensitive fluorescence detection of biomolecules.
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(EBR PG) solution. As a result, Al patterns are formed and connected with each other with Al lines, as shown in panel c of Schematic 1S of the Supporting Information. The measured surface roughness of the lithographically patterned Al thin film is in the range of 6−12 nm, which is smooth enough for carrying out the anodization process and forming NAO. Then, a one-step anodization process is performed to form NAO. Specifically, the samples were anodized in 0.3 M oxalic acid (H2C2O4) at a voltage of 50 V and a temperature of 2 °C for 40− 50 min through the copper clips, as shown in panels d and e of Schematic 1S of the Supporting Information. After these steps, all of the samples are rinsed rigorously using DI water before the experiments. Fluorescence Dyes and Biomolecules and Experimental Procedures. Fluorescent dyes Calceim AM (Sigma, Inc.), Rhodamine 6G (R6G, Lightning Powder, Inc.), fluorescein sodium salt (FSS, Sigma, Inc.), fluorescein isothiocyanate (FITC, Sigma, Inc.), and bovine serum albumin labeled with FITC (FITC−BSA, Sigma, Inc.) were used for the technical demonstrations. Solutions of fluorescent dyes and FITC−BSA are uniformly coated on the NAO substrates and ITO glass substrates. The unbound dyes or proteins were washed away gently using phosphate-buffered saline (PBS) and DI water. The fluorescent images are taken after the solutions on the substrates become dry. Fluorescence Detection, Imaging, and Analysis. All of the fluorescent images have been taken using a fluorescence microscope equipped with a mercury arc lamp source (Olympus, Inc.), which has the following filter sets: FITC (excitation filter, 475−490 nm; barrier filter, 500−540 nm) and TRITC (excitation filter, 545−565 nm; barrier filter, 580−620 nm). Specifically, for FSS, Calceim AM, FITC, and FITC−BSA, the FITC filter set is used for fluorescence measurement. The excitation optical spectrum is from 475 to 490 nm. While for R6G, the TRITC filter set is used and the excitation optical spectrum is from 545 to 565 nm. In this paper, the fluorescence enhancement is estimated and determined by analyzing the fluorescence images using the Imaging Processing toolbox in MatLab.37 A MatLab program has been written to read the files of fluorescence images, which are then converted to gray-scale images from the color images. A horizontal cutline was obtained through the fluorescence image, and the corresponding intensity was obtained and then plotted. To obtain accurate results, the fluorescence images of bare ITO glass and bare NAO have been
EXPERIMENTAL SECTION
Fabrication of the Micropatterned NAO. The basic process is illustrated in Schematic 1S of the Supporting Information. Briefly, an indium tin oxide (ITO) glass substrate is first cleaned by deionized (DI) water, acetone, ethanol, and then DI water for 20 min with each solution. Then, a lift-off process is carried out to obtain Al micropatterns. Specifically, a layer of LOR7b (Microchem, Inc.) followed by a layer of Shipley PR1813 is spun on the substrate and patterned using optical lithography. Using electron-beam evaporation, 2.2 μm of Al is deposited on the substrate with 10 nm of Ti as an adhesion layer. Thereafter, the photoresist is removed by immersing the whole wafer into an Positive Radiation Resist Edge Bead Remover 2440
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Figure 2. (a) AFM image of the NAO. (b) SEM image of the NAO on the glass substrate. (c) SEM image of the substrate after etching the NAO.18
Figure 3. (a) Schematic showing the three different fluorescent dyes or biomolecules labeled with FITC (e.g., FITC−BSA) on the NAO micropatterns or glass. The optical micrographs and the corresponding fluorescence images of three different dyes and FITC−BSA: (b) FSS, (c) Calcein AM, (d) FITC−BSA, (e) R6G, (f) optical and corresponding fluorescence image of R6G on the micropatterned Al, and (g) optical and corresponding fluorescence image of R6G on the substrate after the NAO micropatterns etched away. The remaining patterns on glass are anodized Ti. The integration time for all images is 200 ms. measured and used as references. All results have been corrected by subtracting the fluorescence images of the bare ITO/NAO substrates.
The atomic force microscopy (AFM) image of the NAO is shown in Figure 2a. AFM measurements reveal that the surface roughness of the NAO patterns is typically less than 85 nm, even though it contains many nanoscale grains of aluminum oxide, and the surface of the NAO appears quite rough, as shown in panels d-2 and d-3 of Figure 1 and Figure 2b. The NAO is etched away by immersing the NAO substrate in a mixture solution of phosphoric acid (0.4 M) and chromic acid (0.2 M) at 65 °C overnight, followed by rigorous DI water rinse. While the aluminum oxide nanoscale domains totally disappear, some nanopatterns still remain on the glass substrate, as shown in Figure 2c, which are anodized Ti.18 Clearly, the surface of the substrate after the NAO being etched is totally different from that of the NAO in Figure 2b. Fluorescence Detection and Imaging with Micropatterned NAO. The optical micrographs and the corresponding fluorescence images of three fluorophores FSS, R6G, and Calceim AM and FITC−BSA on the micropatterned NAO substrates are given in Figure 3. Calcein AM is widely used in biology to monitor the viability of cells and for short-term labeling of cells because it can be transported through the
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RESULTS AND DISCUSSION Micropatterned NAO. The optical and scanning electron microscopy (SEM) images of several different shapes of the fabricated NAO micropatterns are given in panels a−c of Figure 1. It has been demonstrated that a variety of shapes of the NAO micropatterns can be routinely fabricated from as small as several micrometers to several hundred micrometers with high yield by combining a lift-off process with a one-step anodization process, which is simple, time-efficient, and cost-effective, as described in detail in the Experimental Section, and the sketch of the fabrication process flow is given in the Supporting Information. As seen clearly from Figure 1d, the nanostructures (i.e., nanopores) are formed and distributed among the nanoscale grains inside the NAO micropatterns. The typical size of the nanopores inside the NAO patterns for our experiments is in the range of 10−30 nm. It should be noted that the size of the nanopores and the distance among them can be readily tuned by changing the experimental parameters.19 2441
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Figure 4. Representative optical, fluorescence images and the relative fluorescence intensity of FITC−BSA with different concentrations along the cutlines: (a) 200 μg/mL and (b) 10 μg/mL. The width of the NAO lines is ∼10 μm. The integration time for all images is 200 ms.
fluorescent dyes (i.e., FITC) can be evaluated. Experiments found that the fluorescence signals of these two samples (FITC and FITC−BSA), applied on two identical NAO substrates, show little difference, suggesting that, as far as the fluorescent dyes are in the range of 14 nm, the fluorescence enhancement from the NAO essentially remains the same. Second, the fluorescence signals of FITC and FITC−BSA on poly(methyl methacrylate) (PMMA)-coated NAO substrates, with PMMA thickness from 50 to 100 nm, have also been measured. Experiments found that the fluorescence images/signals decrease with the thickness of PMMA, but the enhancement still can be observed clearly compared to the same amount of FITC and FITC−BSA on the glass substrate. However, it has been observed that, when the thickness of PMMA increases to ∼100 nm, the fluorescence signals of both FITC and FITC− BSA would show negligible enhancement, which might be due to the ∼100 nm penetration depth of the evanescent electrical field from the surface of the NAO.25,26 The fluorescence enhancement capability of the NAO substrate has been estimated using FITC−BSA as a model and by comparing the NAO substrate to the glass substrate. To this end, FITC−BSA with a series of different concentrations has been applied on the substrates containing the micropatterned NAO. The fluorescence images of two different concentrations (200 and 10 μg/mL) are given in panels a and b of Figure 4. To obtain these images, the integration time is chosen to be 200 ms to minimize the background noise from both glass substrates and the NAO substrates, so that it is easy and accurate to estimate the enhancement factor. As expected, the fluorescence intensity increases with the concentration of the applied FITC−BSA. Using the imaging process program in MatLab, as described in the Experimental Section, the relative fluorescence intensities for both cases are plotted along the cutlines shown in the fluorescence images in panels a and b of Figure 4 after being subtracted by the fluorescence noise signals from the blank glass and NAO substrates, respectively. Again, the fluorescence intensity profiles are identical to the profiles of the NAO lines on the substrate. Given the near to zero intensity of fluorescence signals from the FITC−BSA-coated glass substrate, the fluorescence intensity from the micropatterned NAO is estimated to be at least in the range of 2−3 orders of magnitude larger over that on the glass substrate, which is comparable to other reported metal-oxide-based fluorescence enhancement platforms.7 The lowest detection limit, defined as the concentration at which the signal intensity is 3 times larger than that of the noise from the background, is a
cellular membrane into live cells.20−22 FITC−BSA has been used for a variety of biological applications, such as model drugs for achieving the visualization of the protein localization within the microparticle.23 The detailed experimental procedure is described in the Experimental Section. As seen in panels b−e of Figure 3, the fluorescence images for these dyes and FITC− BSA have been significantly enhanced if they are adsorbed on the micropatterned NAO compared to those adsorbed directly on the glass substrate. Specifically, by examining the optical micrographs of the patterns of the NAO, the corresponding fluorescence images have identical patterns of the NAO, indicating that only the NAO can enhance the fluorescence signals. The glass shows no enhancement capability of the dyes or FITC−BSA. To further validate that the fluorescence enhancement only comes from the NAO patterns, two types of control experiments have been performed from panels f and g of Figure 3. One control experiment is to examine if the aluminum micropatterns can enhance the fluorescence signals of R6G. The other is to examine if the fluorescence signals of R6G can be enhanced by the substrate after the NAO has been etched away. The optical micrographs and the corresponding fluorescence images after coating R6G on the substrates are shown in panels f and g of Figure 3. It is clearly shown that, using a substrate with aluminum micropatterns in Figure 3f or a substrate after the NAO was etched in Figure 3g, no fluorescence enhancement has been observed. In contrast, the fluorescence signals of R6G with the same amount being applied on the NAO micropatterns in Figure 3e are significantly enhanced. In addition, the same phenomena of the fluorescence signals and images have been observed for FSS, Calcein AM, and FITC−BSA, which are not shown here. Experiments have been carried out to examine if the fluorescence enhancement is related to the distance between the NAO surface and the fluorescent dyes. First, the fluorescence signals of FITC and FITC−BSA on the NAO substrates have been measured. For valid comparison, the same amount of FITC should be available from both samples. To this end, the ratio of the mass of FITC and the mass of FITC−BSA should be about 1−20 to ensure that the same amount of FITC is available for estimation. While FITC is directly attached to the surface of the NAO, the majority of the FITC in FITC− BSA is not directly but should be only in close proximity to the surface of the NAO because it is bound to BSA of dimensions of 14 nm × 4 nm × 4 nm.24 Hence, the gap effect on the fluorescence signals between the NAO surface and the 2442
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Figure 5. Self-quenching effect analysis: (a) optical micrograph of the substrate coated with Calcein AM without rinsing, (b) corresponding fluorescence image of the same substrate without rinsing, and (c) corresponding fluorescence image of the substrate after rinsing. The integration time for all images is 200 ms.
Figure 6. Stability experiments: optical micrographs and the corresponding fluorescence images of the micropatterned NAO (a) freshly coated with FSS, (b) 1 month later, and (c) measured fluorescence signals during 10 cycles of reusing the same NAO substrate. The width of the lines of NAO is ∼10 μm. The integration time for all images is 200 ms.
obtain optimum fluorescence signals, even though it is coated on the NAO substrates. Stability of the NAO Substrates. The stability of the NAO substrates for fluorescence enhancement has been examined using two types of experiments. First, the measurements of the fluorescence images/signals of the same substrates immediately after the FSS is applied and 1 month later have been carried out. It reveals that fluorescence signals of the substrate remain essentially the same, as shown in panels a and b of Figure 6. Second, the same substrate has been reused up to 10 times to check its stability for fluorescence enhancement. In this case, a series of 10 successive cycles of fluorescent dye attachment, rinsing, fluorescence detection, and rinsing has been carried out. The fluorescence intensity normalized to the one from the fresh substrate is shown in Figure 6c. The fluorescence intensity decreases after 10 cycles to about 85%, indicating the good stability of the NAO substrates.8 While the physical mechanism of the NAO substrate for fluorescence enhancement requires further studies, on the basis of the previous research by other groups and our experiments, this enhancement mechanism is probably mainly related to the following reasons. First, optical scattering of the NAO may play a very important role in the fluorescence enhancement. Specifically, on the basis of the Mie scattering theory, the scattering property of the NAO is related to the nanopore size, the distance among pores, the excitation optical wavelength, and the oxygen-deficient defects in NAO, which have been confirmed by the photoluminescence measurements of the blank NAO substrates.28−33 The surface scattering effects of the NAO cause the redistribution of the electromagnetic fields with high surface intensities, resulting in the enhanced fluorescence.34,35 Second, the fluorescence enhancement may also result from the evanescent electrical field from the surface of the nanoscale NAO grains, similar to other reported metal oxide nanoscale materials.7−9,18 This assumption has been
more useful parameter for fluorescence-based assays. In these experiments, the integration time increases to 1 s for obtaining the fluorescence images. Experiments found that 0.1 μg/mL (∼1.4 nM) of FITC−BSA can be readily detected using a conventional fluorescence microscope (Olympus, Inc.). While the fluorescence signals of the fluorophores on the NAO increase with their concentrations, the concentration of fluorophores cannot be too large. In other words, the fluorophores on the NAO cannot be too densely packed together; otherwise, the fluorescence signals will be quenched because of the short-range interaction among them.27 The fluorescence self-quenching effect has been analyzed on the micropatterned NAO substrates to explore the optimum fluorescence enhancement conditions. To this end, a drop of the fluorescent dye Calcein AM is applied on the micropatterned NAO region of the substrate, which can be clearly observed optically in the optical micrograph of the substrate in Figure 5a. After a certain time of incubation, the fluorescence images were obtained, as shown in Figure 5b. Some level of fluorescence signals on the NAO can still be observed in comparison to that on the glass. Intentionally, another drop of Calcein AM of high concentration is also deposited on the glass region of the substrate, as shown in Figure 5a, and its fluorescence image can be observed in Figure 5b as well. Thereafter, the same substrate is rinsed with DI water gently to remove the extra Calcein AM not tightly bound to the NAO and the glass substrate. Then, the new fluorescence images were obtained using the same exposure time (200 ms) by the fluorescence microscope. As shown in Figure 5c, the fluorescence images become significantly stronger on the NAO micropatterns than the glass substrate. In contrast, the fluorescence image of the drop of the Calcein AM on the glass region of the substrate becomes essentially negligible. These experiments indicate that the Calcein AM experiences selfquenching at high concentrations and has to be diluted to 2443
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validated by the aforementioned experiments to evaluate the effect on the fluorescence signals from the gap between the surface of the NAO and the fluorescent dyes. As observed experimentally, the significant enhancement of the fluorescence occurs only in the range of 100 nm from the surface of the NAO substrates. At and beyond 100 nm from the surface of the NAO, the fluorescence enhancement becomes significantly weak. It should be noted that, different from noble metal (i.e., Au and Ag) evanescent field-based fluorescence enhancement technology, such as total internal reflection fluorescence microscopy,25 no dielectric layer is needed to separate the fluorescent dyes from the NAO substrates to avoid the quenching effect. Instead, the fluorescent dyes can be directly deposited on the NAO substrates, thereby significantly simplifying the experimental procedure. Another important experimental observation is that the enhancement in the signalto-noise ratio (SNR), a key performance metric and an important parameter for the detection limit of the NAO substrates, has some difference for different fluorescent dyes (such as R6G and FSS) but in a similar order.18 This difference might result from the following reasons: (1) different fluorescent dyes have different quantum efficiency/yield,36 and (2) one type of NAO substrate is more favorable for one specific fluorescent dye than the other because of its specific optical properties, which are determined by the nanopore size and interspacing and the anodization process.29−33 This observation indicates the possibility to optimize the enhancement in the SNR for one specific fluorescent dye using a proper NAO substrate with the optimized optical properties, which can be achieved by tuning the nanopore size and interspacing in NAO.29−33 The favorable features of the NAO micropatterns for fluorescence enhancement therefore can be summarized as follows. First, the NAO micropatterns can be easily fabricated by combining a lift-off process and a one-step anodization. Hence, the fluorescence enhancement technical platform can be made rapidly and very cost-effectively. Second, the fluorescent dyes or labeled biomolecules can be directly attached to the surface of the NAO to achieve fluorescence enhancement. Different from the MEF, a thin layer (i.e., several nanometers) of dielectric material is not required as a spacer to separate the NAO surface and the fluorophores. Third, because the fabrication process is compatible with standard lithographybased microfabrication, the numbers of the arrayed NAO micropatterns can be easily scalable and the shapes of NAO micropatterns can be readily varied or modified, suggesting the simplicity for integrating NAO micropatterns into microdevices or microfluidic devices for fluorescence-based bioassay applications, such as microarray applications for highthroughput DNA sequence and single-cell imaging.
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ASSOCIATED CONTENT
S Supporting Information *
Fabrication process flow of micropatterned NAO (Schematic 1S). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The research is supported in part by the National Science Foundation (NSF) Pilot Funding for New Research (Pfund), Louisiana, 2012.
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REFERENCES
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CONCLUSION In summary, a new type of fluorescence enhancement platform micropatterned NAO substrate has been developed, and its capability to enhance the fluorescence signals of the fluorophores and the labeled biomolecules has been demonstrated. The micropatterned NAO substrate can enhance fluorescence signals up to 2 or 3 orders of magnitude compared to the glass substrate with very good stability. Given its simple fabrication process and integrability with microdevices or microfluidics, this technology offers a new avenue for fluorescence-based bioassay and analysis. 2444
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dx.doi.org/10.1021/la304833u | Langmuir 2013, 29, 2439−2445
