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Droplet Enhanced Fluorescence (DEF) for Ultra-sensitive Detection using Inkjet Hulie Zeng, Daisuke Katagiri, Taisuke Ogino, Hizuru Nakajima, Shungo Kato, and Katsumi Uchiyama Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01566 • Publication Date (Web): 10 Jun 2016 Downloaded from http://pubs.acs.org on June 10, 2016
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Analytical Chemistry
Droplet Enhanced Fluorescence (DEF) for Ultra-sensitive Detection using Inkjet Hulie Zeng*, Daisuke Katagiri, Taisuke Ogino, Hizuru Nakajima, Shungo Kato, Katsumi Uchiyama* Department of Applied Chemistry, Graduate School of Urban Environmental Sciences, Tokyo Metropolitan University, 1-1 Minamiohsawa, Hachioji, Tokyo 192-0397, Japan ABSTRACT: A fluorescence enhanced phenomenon was found within a micrometer-sized liquid droplet, and it was adopted to construct droplet enhanced fluorescence (DEF) for ultra-sensitive fluorescence detection. In this paper, an inkjet was utilized to eject perfect spherical droplets to construct a micro-spherical resonator, and to develop a DEF system. It was utilized to implement ultra-sensitive fluorescence detection in a liquid specimen with a volume of several microliters. The DEF detection of fluorescent molecules, fluorescein sodium, was used as a model to validate the proposed enhanced fluorescence detection method. A low limit of detection (LOD) for fluorescein sodium of 124 pM was obtained. The sensitive detection of single stranded DNA (ssDNA) was experimentally completed, with a wide range of linearity with a LOD of 312 pM. The proposed mechanism/technique can be used as an ultra-sensitive detection technique for analyzing microliters of liquid samples.
Interface enhanced fluorescence is a propagating wave that emerges in the presence of an interface, therefore, it can also be considered to be an evanescent wave. It would be invisible in the absence of an interface, and it is generally converted into a propagating plane wave at the interface 1,2. Generally, the presence of an interface would significantly modify the angular emission of a fluorophore when the refractive indexes of two transparent phases at interface are different. The fluorescence emitted at the interface prefers to be oriented with the phase with a high refractive index as an angular emission3, which could be utilized to improve the efficiency of fluorescence collection. Fluorescence detection can be improved by raising the contrast by eliminating the surrounding fluorescence, decreasing Rayleigh scattering4, and enhancing the fluorescent light collection5. In addition, the enhanced fluorescence can be further intensified by an surficial plasma at the interface, which excites the closed fluorophores or dipoles, thus enhancing the interfacial fluorescence6-10. Investigations of plasmatic enhanced fluorescence have mainly focused on metal surface enhanced fluorescence11-20. The improved local electromagnetic field at an interface was confirmed to greatly excite neighboring atoms, molecules and nanomaterials thus enhancing fluorescence, Raleigh scatting and Raman scatting21. If the interface is located on a smaller surface22, the interfacial fluorescence would be further enhanced by the additionally surficial activation energy of quantum dots23,24, nanotubes25, nanoparticles26-28, nanoclusters29 and 2 dimensional nanoparticle sheets30 as well. However, our current knowledge of liquid interface enhanced fluorescence is limited because of difficulties related to observing the liquidliquid interface. On the other hand, whispering gallery mode (WGM) optical resonators have considerable potential for use in the fabrication of monochromatic laser, because of its high quality factor (Q)31. WGM optical microresonators have been developed for dielectric spheres32, 33, toroids34, 35 and disks36 to obtain a high value of Q to confine the wave energy inside the microcavity. The silica mi-
crotoroid was developed to function as a high-efficiency wavelength tunable laser35, and appears to have great potential for use in the area of single molecular detection33, 37. Regarding the propagation of energy in a liquid microsphere, dye-doped droplets in air were reported to result in the laser emission, and were observed to highlight fluorescence at a liquid-air interface38. A liquid-liquid droplet was also proposed for fabricating a droplet dye laser39, even wavelength tunable droplet laser40 profiting from the high Q values of WGM droplets and the enhanced fluorescence yields of dielectric fluorophores41. To exploit the potential of liquid WGM microsphere in microanalysis, we simply generated liquid droplets via a sophistic inkjet technique to construct the spherical liquid WGM micro-resonator to obtain DEF, and explored its great potential in the ultrasensitive measurement of fluorescent molecules in a tiny volume of liquid specimen. Actually, the inkjet have great potential for use in automating micro-analytical instrumentation, and have been explored to accurately inject liquid samples for gas chromatography (GC)42, capillary electrophoresis (CE)43, and in automated enzyme-linked immunosorbent assays (ELISA)44. It also has been developed to generate uniform liquid-liquid droplets with a high degree of precision45. In this paper, the fluorescence enhancement due to a droplet with a higher refractive index in a liquid with a lower refractive index was examined, and was utilize to implement ultra-sensitive fluorescence detection. An inkjet was adopted to construct the spherical droplet microcavity to obtain a WGM microresonator to achieve the sensitive fluorescence detection of microliter-sized volume of liquid samples. The DEF system consisted of an in-house built laser-induced fluorescence (LIF) detection system for the detection of droplets, and a liquid droplet generating system for the repeated generation of monodisperse liquid-liquid droplets, as show in Figure1.
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The target sample solution could be simply loaded into a capillary by capillary force. The fully loaded liquid in an erect square capillary supplied a convex meniscus at the top that permits the tip of inkjet to make contact with the nozzle, so that the liquid-liquid droplet can be freely generated at the top of capillary to construct the droplet WGM microresonator as Figure 1 shown. The generated liquid droplet, in an immiscible liquid would drop along the erect capillary with gravity as the driving force. The droplet can be controlled just at the center at the top of capillary without it touching or being suspended on the well of capillary (see extended video) with an X-Y stage holder. The fluorescence signal of a single droplet in less than a hundred micrometers could be collected by the PMT through a 200 µm slit that was set at a landscape orientation when the generated droplet is falling through the square capillary, as shown in the right insert in Figure 1.
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The generation of liquid-liquid droplets by an inkjet results in the formation of a perfect spherical WGM microresonator to propagate an optical wave when the refractive index of the droplet phase is higher than that of the continuous phase. In this case, either the exciting laser or the excited fluorescence would proceed via the most probable multi-reflection within the droplet with a higher refractive index based on the Fresnel equations in Figure 2a. The generated benzyl benzonate droplet whose refractive index is 1.56 in water and the aqueous solution whose refractive index is 1.33 match the above essential requirement for enhancing the fluorescence. The fluorophores within the droplet and at the interface could both be most probably multi-excited, resulting in an enhanced fluorescence. Therefore, the extremely high fluorescent intensity of equilibrated benzyl benzonate droplet contrasting with the background fluorescence of 80 µM of aqueous fluorescein sodium solution was obtained as shown in Figure 2b. The drastic fluorescent enhancement in the benzyl benzonate droplet was not due to increasing the concentration of fluorescein sodium as shown in Figure 3a. In fact, the obvious enriching of fluorescein sodium in the benzyl benzonate droplet cannot be observed in a fluorescein sodium solution at any concentration as shown in Figure 3b. Additionally, we also cannot assume that enhanced fluorescence is due to an adsorption of fluorescein sodium at the interface, because the molecule of fluorescein sodium does not have any surfactivity. Therefore, we deduced that the enhanced fluorescence in droplet is due to the droplet mode microresonator, which we refer to as droplet enhanced fluorescence (DEF).
Figure 1: Set up for the fluorescent detection and generation of droplets in DEF.
Figure 3: Fluorescence spectra of 10 µM fluorescein sodium in water and in benzyl benzonate (a) and distributions of fluorescein sodium in a water phase and in a benzyl benzonate phase. The exciting wavelength was 496 nm.
Figure 2: Principle of droplet enhanced fluorescence (a), and benzyl benzonate DEF in 80 µM aqueous fluorescein sodium solution in 500 µm square capillary (b).
Theoretically, the emission rate could be enhanced when the emission frequency corresponds with the cavity resonance41. The micrometer sized droplet functions as a microresonator to propagate the emission, and improves the fluorescence yield, which can be attributed to a net increase in the fluorescence decay rate. Ultrasensitive fluorescence detection is then achieved via the development of DEF by the inkjet described herein. In addition, the existing of droplet microsphere improves the efficiency of collection of the emitted fluorescence by two effects: (i) a local intensity enhancement due to an extra focusing effect; (ii) an improved
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Analytical Chemistry
fluorescence detection efficiency related to a better collection of the emitted fluorescence at high incidence angles33. Additionally, the generation of a monodisperse liquid-liquid droplet with a high precision by the inkjet45 also ensures that the DEF system operates with a high degree of precision. The flexibility of the diameter of the generated monodisperse droplets through the controlling of driving voltage and pulse width exerted on the pizeo slide in the above inkjet will supply the flexible conditions needed for DEF in Figure S1. In this experiment, the stable generation of an 80 µm of monodisperse benzyl benzonate droplet with a high level of precision was obtained at a driving voltage of 40 V and a driving pulse width of 20 µs.
Table 1. Comparison of LIF detection and DEF* LIF**
DEF
LOD(pM)
577
124
RSD (%)
0.84~5.57
0.73~5.42
0.9576
0.9936
Linearity(R2)
* Five parallel measurements were counted for a sample. ** Signal of LIF detection was obtained through 500 µm square capillary at the same level of laser radiation.
Generally, the distribution of fluorescent molecules in a pure benzyl benzonate droplet is nearly zero, when it is just generated and located at the top of capillary at the beginning in shown in Figure 1. The immergence of fluorescent molecules in the benzyl benzonate droplet trends to gradually equilibrate with the diffusion of the fluorescent molecules from the aqueous solution to the droplet phase, as shown in Figure 4a. Fluorescein sodium was used as a model molecule to search for the point of the equilibrium for the optimum detection point in the DEF detection system, and the appropriate detection point for the proposed DEF system was confirmed to be at 3 cm in Figure 4b, therefore, the least volume of liquid sample needed is around 7.5 µL, as calculated based on the optimized detection point of the square capillary with sides of 500 µm. The time required to complete a measurement was as little as 65s, as shown in Figure 4b.
To explore the application of DEF for a wide range of target analytes, the measurement of ssDNA was investigated for the case of circulating cell-free DNA (cfDNA) in a clinical diagnosis. Deox-
To investigate the possibility of the quantitative analysis of fluorophores, we carried out measurements of fluorescein sodium by DEF and classical LIF through a 500 µm square capillary. The findings are compared in Table 1. A lower LOD and a higher precision were found for the DEF detection compared with that for LIF detection. We believe DEF is a detection technique that can overcome the limitations associated with ultra-high sensitivity in microanalysis.
The distribution of GelGreen labeled ssDNA would not be enriched in the benzyl benzonate droplet phase, similar to the case of fluorescein sodium as shown in Figure S3, but the natural characteristics of DNA would tend make it adsorbed at the interface4749 . The ssDNA might be concentrated further at the interface of the droplet to further enhance the fluorescence via increasing the amount of fluorophores close to the outer shell of the droplet, as shown in Figure 5a. The DEF technique presents an opportunity to sensitively detect biomacromolecules, based on their tendency gather at the interface and thus further enhance the fluorescence of the droplet microresonator.
yribonucleic acid, single stranded, from calf thymus (~50kb) was selected as the model ssDNA to explore the potential of DEF for the analysis of such a molecule. First, ssDNA solutions were prepared in 0.1 M PBS buffer (pH 7.4), and labeled by a 10000 times diluted GelGreen nucleic acid gel stain to produce fluorescence emission. Since the original fluorescence of the free GelGreen molecule, AOAO-13, is negligible46, we assumed that any detectable fluorescence can be attributed to the presence of GelGreen labeled ssDNA, therefore, it would be feasible to quantify ssDNA by fluorescence labeling as shown in Figure S2.
Figure 4: The immergence of the fluorescent molecules into a droplet during it passes down the capillary (a) and the optimization of the detection point of the DEF system using a 15 µM fluorescein sodium solution.
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great potential for using DEF for the sensitive and precise analysis using extremely small volumes of sample, as shown in Table 2. In summary, a liquid droplet with a higher refractive index emerged in a liquid with a lower refractive index, resulting in an enhanced fluorescence phenomenon due to the WGM effect in the droplet microresonator. The inkjet was adopted for use in the DEF system premitting the super sensitive fluorescence detection of a liquid sample in the microliter range. The constructed DEF system was used as a model for WGM enhanced fluorescence in a liquid microresonator to increase the fluorescence yield by the propagation of fluorescence emission, which has the potential to overcome the limitations associated with sample volume on the sensitivity of the method. Moreover, the aggregation of biomolecules, such as DNA molecules, on the surface further improved the sensitivity of DEF. The method has great potential for use in the sensitive measurement of liquid bio-specimens in the microliter range. ASSOCIATED CONTENT
Supporting Information The Supporting Information is available free of charge via the ACS Publications website at http://pubs.acs.org. Figure 5: DEF of GelGreen labeled ssDNA (a) and calibration curve of ssDNA. Table 2. Comparison of the detection of ssDNA by DEF and conventional fluorimetric method*
Consumption reagent (nL)
of
Volume of sample (µL) LOD(pM) Linear range(nM) Linearity (R2) RSD (%) Measurement time(min)
AUTHOR INFORMATION Corresponding Author *
[email protected],
[email protected] Fluorimetric method
LIF**
DEF
0
0
1.34
5000~10000
250
7.5
The authors acknowledge the financial support to Dr. H. L. Zeng from Tokyo Metropolitan University and Tokyo government.
155
5901
312
0~2.0
0~10
0~10 and more
0.9946
0.9279
0.9958
0.87~9.76
0.94~10.04
3.25~4.72
5
1
1~2
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* Five parallel measurements were counted for a sample. ** Signal of LIF detection was obtained through 500 µm square capillary at the same level of laser radiation.
The ultra-sensitive quantitative measurement of ssDNA was accomplished and the results are shown in Figure 5b, and an LOD of 312 fM with a wide linearity was achieved. Compared with the conventional fluorimetric method in which the sample is directly measured by a spectrofluorophotometer and the LIF method, the analysis of ssDNA implemented by the proposed DEF system showed nearly the same levels of sensitivity as that for the traditional fluorimetric method, but the sample volume was reduced by 1/1333. The experimental data demonstrates the
ACKNOWLEDGMENT
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