Sensors and Materials, Vol. 14, No. 6 (2002) 293-308
MYUTokyo
S &M 0491

Encapsulation and Stability Properties of
Nanoengineered Polyelectrolyte Capsules for
Use as Fluorescent Sensors
Ted A. Duchesne 1, J. Quincy Brown1, Kyle B. Guice 2, Yuri M. Lvov andMichael J. McShane*1
Institute for Micromanufacturing, LouisianaTech University,
911 Hergot Avenue, PO Box 10137, Ruston, LA 71272, U.S.A.
1
Biomedical Engineering Program, 2Chemical Engineering Program(Received November 5, 2001; accepted February 5, 2002)
Key words:

electrolytes

fluorescent sensors, layer-by-layer electrostatic self assembly, nanoparticles, poly­This is the first report about a novel fluorescence sensor technology based on hollow
micro- and nanoscale polyelectrolyte capsules. The nanostructured shells were con­structed using the electrostatic layer-by-layer assembly process to deposit multilayer
polyion films onto rnicrotemplates (melamine formaldehyde microspheres). The latex
cores were subsequently dissolved and removed, leaving hollow shells. The capsules werethen loaded with a model fluorescent assay consisting of a sodium-sensitive dye and areference fluorophore. Fluorescence spectroscopy was used to analyze properties of thecapsules with respect to their potential application as biosensors. The results show thatmultiple dye molecules can be introduced into the interior of the capsules with excellent
control over relative levels, and the capsules retain >99% of fluorescence during 30 days ofstorage in a buffer. The findings also demonstrate that the capsules are mechanicallyrobust, and only extremes in solvent pH cause significant leaching of fluorophores from theinterior of the shells. Finally, results from sodium sensitivity experiments suggest that
capsules have excellent potential for use as sensors, with a highly linear response over abroad range (0-100 mM).

1.

Introduction

Chemical sensors are important for monitoring of chemical processes and biologicalsystems.<lJ In recent years there has been an increased interest among researchers in*Corresponding author, e-mail address: mcshane@coes.latech.edu
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developing novel, improved approaches for determining biochemical concentrations.<2l
While modern biosensors provide a basic ability to quantitatively measure chemical
substances, many suffer from an inability to provide adequate specificity, spatial resolu­
tion, and real-time sensing capability. The use of many such sensors is further hindered byassociated damage to biological tissues due to implantation of large sensors, analyte
consumption, performance degradation over time, or repeated invasive use. <2-34i
Optical sensing techniques have gained popularity as alternatives to the more common
electrochemical methods. Advantages of these sensors include minimal consumption ofanalytes, potential for real-time monitoring, insensitivity of the signal to electrical interfer­ence, electrical isolation, and high sensitivity to analytes.<5•6l Specifically, fluorescence
spectroscopy and microscopy have been utilized for the real-time, specific and extremely
sensitive monitoring of biochemicals, pH, and voltage.
The most common use of fluorescence in biological measurements is the injection of afluorescent indicator dye directly into a cell or tissue sample. In principle, these fluores­
cent indicators operate based on the change in excitation or emission properties (intensity,
spectral shape, and/or lifetime ) due to binding with analyte molecules, quenching offluorescence by the analyte molecules, or fluorescent resonance energy transfer (FRET). <7,8l
Disadvantages of these methods include cytotoxicity of the assay chemistry, intracellularsequestration, protein binding, and diffusion of dye from the area of interest, all of which
affect the surrounding tissues as well as the experimental results .<8l Thus, efforts toimmobilize the sensing chemistry into a biocompatible probe configuration have been
pursued.
The majority of researchers in the field of fluorescence sensing have studied various
configurations for fiber-optic probe-based sensors.< 1 •6 •9) A pH sensor was the first fiber­
optic-based device designed specifically for physiological useYOl Since then, a number ofnovel fiber-optic, integrated probe systems have been developed to detect importantanalytes such as glucose, oxygen, and carbon dioxide.0· 1 1• 12l While these fiber-optic probe­based sensors offer high strength and stability, they still require the physical connection offibers to the sensing elements and have not found widespread applicationY 3l
Recently th.ere have been attempts to decouple the sensing chemistry from opticalfibers. This capability is important for intracellular measurements and chronically im­planted sensors thatcan be interrogated transdermally. It can also enable fluorescenceimaging, with the physical entrapment of assay molecules to avoid leaching, and the added
potential for protecting the cells from toxic chemicals. In addition, encapsulation ofindicator dyes by biocompatible materials enables sensors to remain unaffected by protefn
adsorption. <8l
Potential solutions to these problems that are currently under investigation involve theuse of fluorescent indicators embedded in polymer or silica matrices,<8• 14--16l and encapsu­
lated by dialysis membranes or liposomesY 7• 18l Advances in polymer and sol-gel systems,
the most intensely investigated, have largely overcome a number of problems in term� ofsensor sensitivity, response time, indicator leaching, and reversibility(8l with recent im­provements in fabrication processesY 5l However, the fluorescence intensity and homoge­neity of particles produced using polymerization is still dependent on the solubility of
assay chemistry in precursor solutions, which often results in poor homogeneity, as well as

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particle-to-particle and batch-to-batch repeatability. In addition, the use of solid polymer
or hydrogel systems alone complicates the use of competitive-binding reactions, which
require free movement of molecules within a restricted range such as is obtained withdialysis membranes. This option, however, while suitable for miniaturization, is not
amenable to efficient fabrication, will have poor mechanical stability and durability, andrequires relatively large end-products that cannot be deployed intracellularlyY 1>
An interesting possibility for improving the current methods is the use of tiny fluid­
filled shells: in the past few years, a new technique for the fabrication of hollow capsules
has been developed to produce tiny shells that can potentially be employed as carriers forfluorescent sensors. The electrostatic layer-by-layer (LBL) assembly technique has been
elaborated to deposit thin layers of charged molecules onto micro- and nanoscale tem­plates, with subsequent removal of the core material to achieve hollow shells.< 19-27J
The method is very versatile, and shell properties can be controlled: inner diameters are
dependent upon the size of templates used for assembly, wall thicknesses can be controlled
precisely to within a few nanometers, and walls may be constructed using a diverse
selection of charged molecules (polyelectrolytes, proteins, dyes, inorganic particles, co­polymers, etc.).<24> The versatility in the construction of these tiny capsules and theircontrol over properties makes them attractive for use in sensor applications.
The aim of this work was to study the potential for real-time sensors based on hollow
polyelectrolyte capsules with specific transport properties. This novel concept was studied
to determine the potential of dye-loaded shells for measurement of chemical concentra­
tions. A model two-component sodium assay was chosen, which included a sodium­
sensitive dye and a reference fluorophore, the latter of which is used for an internal
intensity reference. Sodium was chosen as an initial analyte because of its importance tocellular function, as well as the availability of sodium-specific fluorescent probes.<23 >Using the assay-loaded capsules, three sets of tests were performed. The loading proper­
ties of shells were studied to determine the possibility of simultaneous encapsulation ofmultiple molecules and the controllability of relative amounts of the multiple internalized
species. The fluorescence intensity of loaded shells in suspension was compared as ameasure of signal levels. The stability and robustness of the capsules were tested undermechanical insult (vortexation, sonication, smashing) and pH modulation. Finally, simple
assay response experiments were conducted to confirm that the indicator maintains its
function after encapsulation as well as to determine the potential range and sensitivity
achievable with such sensors.

2.

Materials and Methods

2.1 Reagents

Polyelectrolytes used for the formation of the shells were sodium poly(styrenesulfonate)(PSS, MW-70 k, polyanion) and poly(diallyldimethylammonium) chloride (PDDA,
MW- 100 k, polycation) (Aldrich Chemicals, Milwaukee, WI). The substrate particles
were positively charged, 2.3-µm-diameter melamine formaldehyde (MF) in 10% V/V
solution of KCl (Microparticles GmBH, Germany). The sodium-sensitive dye used was
sodium-binding benzofuran isophthalate (SBFI) and the reference fluorophore was 7-

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methoxycoumarin-3-carboxylic acid (MCA) (Molecular Probes, Eugene, Oregon). The
pH 7.6 (0.02 M) buffer was made with Trizma acid and base (Sigma Chemical, St. Louis,MO).

2.2 Instrumentation

A 1510 Sonicator (Branson, Danbury, CT) and a Genie 2 Vortex (Daigger, Bohemia,
NY) were used for dispersion and suspension of particles in microcentrifuge tubes. A
5404R Centrifuge (Eppendorf, Westbury, NY) was used to separate the particles fromambient solution. A Nitrogen laser (LSI, Franklin, MA) was used as the excitation source
for fluorescence experiments. A fiber-optic 2048-element PDA spectrometer (StellarNet,
Inc, Oldsmar, FL) was connected to a custom cuvette holder using an SMA-terminatedprobe comprising seven (six around one) 400 µm fibers. The data were collected using
vendor-supplied software (SpectraWiz), then imported and analyzed using Microsoft
Excel.

2.3 Spectroscopy

Samples were placed in 1 cm cuvettes and were stirred prior to data collection tomaintain homogeneity. Three replicate scans were taken for each sample so instrumental
variations could be assessed. Spectra were collected using 2000 ms integration time, andfive consecutive scans averaged to produce a single spectrum.

2.4 Shell fabrication

The shells were fabricated using layer-by-layer assembly techniques that have been
described in detail elsewhere_(29-33 l Briefly, polyelectrolytes were mixed with 0.5 M KCl toobtain stock solutions (2 mg/ml PSS, 3 mg/ml PDDA). It is known that ionic strength is adetermining factor in the structure of assembled layers and, for this study, this variable washeld constant by using the same stock solutions of PSS and PDDA for all deposition
reactions. Approximately 100 µL of the MF particle suspension (-109 particles) was
placed in each of four micro-centrifuge tubes; then 1 mL of PSS stock solution was added
to each tube. The particles were vortexed and sonicated for one minute, suspended in the
PSS stock for ten minutes to allow polyelectrolyte adsorption, then centrifuged at 10,000rpm for ten minutes to separate particles from solution. The rinse cycle involved removing
the supernatant, adding 1 mL of Trizma buffer, sonicating, vortexing and centrifuging.
Each batch was rinsed three times for each step. After the third cycle, the supernatant wasremoved, and 1 mL of PDDA stock solution was added to the particles. The particles werevortexed, sonicated and suspended for ten minutes. The three-cycle rinse process was
repeated, leaving the particles with one complete bilayer. This procedure was repeateduntil there was a total of five bilayers on the MF particles.
Once the layering process was complete, the cores were dissolved by placing theparticles in a 0.1 M NaCl solution.c24l The particles were suspended in the NaCl for 24 h.
During this time the MF cores dissolved and diffused out of the shell. The microshells
were centrifuged and rinsed ten times to remove residual NaCl. This process resulted inhollow capsules surrounded by five bilayers of polyelectrolytes_C24l

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2.5 Dye loading experiments

For the capsules to be used as carriers for fluorescence sensor chemistry, a means of
introducing the molecules into the shells is necessary. Initial tests studied diffusion due tothe simplicity of the approach. Individual stock solutions of SBFI (0.05 mg/mL or 55 µM)
and MCA (0.1 mg/mL or 454 µM) were prepared, then a mixture of3 :2:5 (SBFI:MCA: buffer)
was prepared from stock solutions, resulting in a mixture of 0.015 mg/mL (16.5 µM) SBFIand 0.02 mg/mL (91 µM) MCA. Hollow capsules were suspended in 1 mL of the SBFI/MCA solution for a period of 24 h to allow diffusion of dyes through the bilayer
membranes and into the interior of the capsules. The shells were rinsed ten times to remove
the free dye. Figure 1 depicts the steps of capsules formation, from the nanoassembly ofpolyion layers on the particle surface to the loading of dyes.
Control experiments exposing only MF particles (no polyions), MF particles with afive-bilayer polyion coating (MF core intact), and five-bilayer hollow capsules (coredissolved) to the same dye mixture were used to determine whether dyes adsorbed onto
MF, whether they stuck to or became entrapped within polyion films, or whether theydiffused into the interior of polyion shells.
Another essential aspect of carriers for fluorescent indicators is a way to control theamount of material ente1ing the vehicle inte1ior. Because of the need for two-dye systems
in quantitative measurements, some understanding of how to tailor the relative amounts ofthe two molecules loaded into the shells was also important. A series of experiments toe"-.

f;Fig. 1. Steps of capsules fabrication. Layering of polyelectrolytes (a-e) until the desired number ofbilayers are deposited. Once the layering process is complete, the core is dissolved, leaving behind
an empty shell (f). The hollow shells are then loaded with sensor chemistry (g).

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follow the simultaneous diffusion of both indicator and reference molecules over time wasused. After the template particles had been dissolved and capsules isolated, the shells were
loaded with the same 3:2:5 volume ratio (SBFl:MCA:buffer) solution as noted above. To
achieve this, the capsules were suspended in 1 mL of the dye mixture at room temperature.
After the shells had been in the solution for one hour, 100 µL of the suspension wasremoved and capsules were separated and rinsed using five cycles of centrifugation at
10,000 rpm for five minutes. They were then suspended in 0.5 mL of Trizma buffer andfluorescence spectra of the suspension were recorded. After allowing some time (-1 h) forthe capsules to settle to the bottom of the cuvette, the solution was scanned using the fiber
probe with the laser coupled into the central fiber for excitation and the surrounding six
fibers for collection. This allowed excitation of the sample only where the probe wasplaced. The solution was scanned at the top, in the middle, and at the bottom, where theparticles had aggregated. This process was repeated after 2, 3, 4, 5, 6, 9, 12, 18 and 24 h hadelapsed, so loaded shells could be observed after different periods of immersion in the dye
mixture. Spectra of the 3:2:5 SBFI:MCA:buffer mixture used for loading the capsules
were also collected for purposes of comparison.

2.6 Stability testing

Longevity and stability are both desirable qualities of sensors, particularly those forbiomedical applications. Containment of assay chemistry is a critical property that affects
the shelf-life of sensor materials as well as longevity in continuous use. For the hollow
capsules described here, it was important to demonstrate that assay chemistry, once loaded,would be retained inside the shells for a sufficient time period under normal storage
conditions. Furthermore, it is important to know whether the capsule walls lose structure(rupture) when mechanically stressed or in a hostile chemical environment. In this work,
dye-loaded capsules were studied under exposure to different conditions to determine theintegrity of capsule walls and the potential duration of encapsulation. These conditions
included vortexing and sonicating, long-term storage, and suspension in acidic and basic
solutions.
Stability during storage. Long-term storage tests were performed to determine how much
dye leached out of the capsules over a period of one month. Loaded capsules weresuspended in a fresh buffer solution, and baseline fluorescence spectra of the shellsuspension and supernatant (after centrifugation) were collected. The shells were re­suspended in a microcentrifuge tube and placed in the refrigerator for storage. Every otherday, the suspension was centrifuged, supernatant was drawn off and placed in a cuvette,and fluorescence spectra were collected. The supernatant was then placed back into thecentrifuge tube with the capsules and they were re-suspended. The fluorescence intensityof the capsules was measured only on the first and last days of the testing. These steps weretaken to gain some insight into the leaching of dye molecules from the interior of the shells
into the ambient solution.
Stability during Vortexation and Sonication. The purpose of this experiment was todetermine if fluorescence molecules moved out of the capsules during vortexing orsonication of the capsules, due to either destruction of walls or increased permeability. It

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is noteworthy that, as described above, both of these procedures were used throughout thepolyion shell fabrication process as a means of suspending the particles in solution. Afterthe capsules were loaded with dyes, one vial was marked for vortex only; the other forsonicate only. Throughout the rinsing cycles, each vial was either vortexed or sonicated forone minute in accordance with its notation. After ten rinses, the capsules were left
suspended in the pH 7.6 buffer and fluorescence spectra were collected. Then the capsules
were centrifuged, and only the supernatant was scanned,
Stability in Acidic and Basic Environment. Polyioi1 capsules are proposed as a versatiletechnology for encapsulation of fluorescence chemistry. It is also important, then, tounderstand how they respond when placed in a basic or acidic environment. This isbecause many fluorescent indicators are not soluble at neutral pH and thus loading into
capsules would likely require suspension of the capsules in an acidic or basic solution. In
addition, it is possible that capsules might eventually be used for applications that require
operation in a nonneutral medium, for example, as pH sensors. It has aiready been shownthat pH does affect the wall permeability of capsules fabricated using multilayer polyelec­
trolyte films.<2 1> For this work, simple tests were performed to determine the relative
amount of the fluorophores released from the loaded capsules in both acidic and basic
solutions. After the capsules were loaded and rinsed ten times, a pH 9.0 buffer was added
to one vial, and a pH 4.11 buffer was added to another vial. Fluorescence scans of both theparticles and supernatant were performed. The capsules were suspended overnight in theirrespective buffers. After 24 h, fluorescence spectra of shells and supernatant were again
collected.

2.7 Sodium response
A final concern regarding the suitability of the shells as carriers for fluorescence assays
is the ability of small molecules to equilibrate across the capsule walls, and interact with theindicator molecules. Using the sodium assay described above, initial experiments were
performed to demonstrate .the feasibility of capsules loaded with SBFI to respond tochanging sodium levels. A cuvette containing 0.5 mL of loaded capsules suspended in pH
7.6 tris-buffer was placed into the sample holder. A NaCl solution was prepared bydissolving 2.925 g of NaCl in 100 mL of tris-buffer. This NaCl solution was prepared suchthat adding 10 µL of NaCl solution to the 0.5 mL capsules/buffer solution gave a 10 mM
increase in NaCl concentration. The NaCl concentration in the capsules/buffer solution
was increased in 10 mMincrements from 0 mM to 100 mM. At each concentration, thesuspended capsules were excited with the LSI laser, and three scans from the spectrometer
were recorded.

3.

Results and Discussion

3.1 Dye loading experiment
The dye loading experiment was performed in an attempt to understand the propertiesof diffusion of individual dyes into the capsules. Figure 2 shows fluorescence spectra ofcapsules after immersion for 1, 12, and 24 h in a dye mixture. The spectra in Fig. 2 have

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Wavelength (nm)Fig. 2. Intensity (normalized to 518 nm) of capsules suspensions at 1 (thick dashed), 12 (thicksolid), and 24 h (thin solid) during the loading experiment.

rising relative fluorescence at the shorter-wavelength peak, which indicates increasing
encapsulation of MCA in the shells for longer loading times. The data seem to show that
SB.FI enters the shells at a faster rate initially, but the capsules continue to take up MCA
until the fluorescence peak intensity surpasses that from SBFI as the system approaches
equilibrium. This trend is surprising, because the SBFI molecule is almost three times
larger than the MCA molecule. Furthermore, there were many more MCA molecules in
the loading mixture. The observations suggest that neither wall permeability nor concen­
tration gradients determine the transport behavior of the dye molecules to the interior of thecapsules. Further experiments are needed to fully understand the processes involved in dye
uptake, but the intention here was to determine whether the process is truly encapsulation
and whether the results are repeatable.
Control experiments showed no appreciable fluorescence of solid MF particles or MF
particles with five outer polyions bilayers when compared to hollow capsules aftersuspension in the dye mixture. These results indicate that the fluorophores are not simply
adsorbing to the surface of particles or becoming entrapped within the polyion films. The
explanation for dye retention within the capsules after diffusion is still unclear, thoughseveral hypotheses have been developed. These include: 1) dye aggregation in the capsuleinterior, 2) dye molecules complex with remaining fragments MF in the capsule, and 3) alayer of charged dye molecules coats the inner surface after diffusion, resisting outward
diffusion of other molecules by electrostatic repulsion. Polarization experiments showedanisotropy values of 0.6-0.7 for encapsulated dyes, which suggests some binding/immobi­
lization of molecules or high viscosity in the capsule interiors. Future work using confocal
fluorescence microscopy, absorption spectroscopy, and polarization experiments is neededto address this question in more depth. From an engineering standpoint, however, it issufficient to know that the process is repeatable and controllable.

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Figure 3 shows the fluorescence peak ratio (MCA:SBFI) as a function of time ofremoval from the dye solution for the shells during loading. During the first twelve hoursof loading, there was little change in the fluorescence peak ratio of MCA:SBFI. However,there was a significant and steady increase in peak ratio during hours 12-24. The
importance of this behavior is that one can "tune" the relative amount of loading of two
molecules by carefully controlling the immersion time. The curve fit to the data points
suggests that there is an almost exponential increase in peak ratio with immersion time.
Further experiments showed that this curve can be used to select a time that will result in apeak ratio within 10% of that estimated by the equation, if the same dye mixture, shell size,
wall thickness, and wall composition are used.

3.2 Liquid phase vs. encapsulated dyes

It is well known that fluorescent molecules are sensitive to the environment in which
they are placed. It was important to compare emission spectra of capsules to spectra ofdyes in solution, as that is an indication of whether the fluorophores truly migrate to theinterior, adsorb to the surface, or get entrapped in the polyion walls of the shells. In theory,the interior of the polyion shell is similar to the surrounding solution. If this is true, thelocation of emission peaks in spectra collected from capsules should be very similar to
those seen in the loading mixture, unless there is a significant change (e.g., pH shift)encountered in the capsule interior.
Figure 4 contains plots of emission spectra for capsules and a dye mixture used to load
the shells. The data show that the emission peaks of the respective dyes occur at theapproximately the same wavelengths for both liquid phase and encapsulated dyes. The
observed wavelength of MCA emission is approximately 420 nm, and the observed SBFIemission peak occurs at approximately 550 nm. The results show that, other thandifferences in overall intensity and a slight peak shift, the spectra are essentially the same.
The SBFI and MCA emission peaks at approximately the same spectral position in both the
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Time (hrs)Fig. 3. Ratio of fluorescence intensity at peaks (418 nm: 518 nm) over the 24 h period during
immersion in dye solution. Markers show computed ratio values, while the line is an exponential fit.

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Fig. 4. Comparison of normalized emission spectra of dyes in solution (thick solid) to loaded
capsules (thin solid).
capsules and solution phase. These findings indicate that the dyes are most likely diffusing
into the capsules instead of adsorbing to the surfaces, which experience shows would givesignificantly different spectra. It should also be noted that different batches of SBFI had to
be used throughout the experiments, and significant batch-to-batch variability in propertiesof this particular dye has been previously reported,C34J so shifts in emission properties were
expected even between solution spectra.
Another important characteristic of carriers for optical sensors is the ability to take up
enough of the indicator molecules to produce detectable signals. A desirable property is anemission intensity that exceeds that from dye in solution. The fluorescence obtained from
capsules was compared to the dye mixture used to load the sensors (0.015 mg/mL SBFI and0.02 mg/mL MCA). Figure 5 contains spectra from an experiment that used the same
instrumental setup to record data from both samples. The fluorescence of the loaded
capsules suspension is two orders of magnitude more intense than that of the dyes insolution. This result far exceeded expectations, and bodes well for applications of thefluorescent capsules to low-light situation. An interesting point is that during the loading
process, the concentration of dyes does not equilibrate between the interior and exterior of
the shells. If equilibrium were reached, the suspension of capsules could never evenapproach the intensity of solution, because the volume of dye and, hence, the number offluorophores would always be smaller.

3.3 Stability tests

Long-Term Stability. The long-term stability experiment was performed in order to test
the ability of the capsules to retain the encapsuhited dyes over a period of one month.
Figure 6 shows spectra of capsules on the last day of the study together with thefluorescence of supernatant on selected days throughout the period. It is noted that thecapsules used in this experiment were loaded over a short time and, therefore, exhibit a303

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Fig. 5. Comparison of relative fluorescence intensity of dye solution (thick solid, 0.015 mg/mL
SBFI and 0.02 mg/mL MCA) to dye-loaded capsules (thin solid).

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Fig. 6. Spectra of capsules and supernatant during the one-month storage period. The more intense
spectra were collected from a capsule suspension at the end of the 30-day experiment. Weaker
spectra that are barely visible �ere collected from supernatant on days 1 (triangle), 9 (diamond), 17(square), and 29 (circle).

larger emission at 510 nm (SBFI) compared with that at 400 nm (MCA); this explains therelationship between Figs. 2 and 6. The supernatant spectra in Fig. 6 are one order of· magnitude weaker in fluorescence in all cases, and can barely be seen on the graph. It isclear from these data that the fluorescence of the capsules was much more intense than eachSensors and Materials, Vol. 14, No. 6 (2002)

304of the supernatant scans taken throughout the month, even at the end of the period. This
demonstrates that the amount of dye leaving the interior of the capsules is at the noise level
when compared with the fluorescence from the capsules.
The relative loss of dye to solution over the one-month storage period was placed on aquantitative scale by computing the change in fluorescence intensity of the supernatant asa percentage of the initial capsule fluorescence at 510 nm. Calculations showed that theloss of dye to the ambient solution results in a fluorescence signal less than 0.1 % of theintensity of the fluorescent capsule suspension. Thus, leaching did not occur significantly
over the testing period. Thus, the contribution of nonencapsulated dye to fluorescencemeasurements will not be appreciable, reducing the likelihood of spurious signals or
widely varying nonspecific background levels of fluorescence.

Mechanical and Chemical Stability. Initial mechanical tests were conducted to discoverwhether processes such as vortexing and sonicating had any adverse effects on thecontainment of the dyes inside the hollow cores. Figure 7 shows spectra from experiments
where loaded capsules were subjected to vortexation and sonication. The spectra aresimilar to those obtained from the storage experiments. Again, the fluorescence intensityfrom the supernatant is extremely small compared to that obtained from the capsules, to thepoint that most spectra cannot even be observed when placed on the same scale as thecapsules. This indicates that very little dye is lost through the shell during these proce­dures. A small increase in leaching is noted when comparing fluorescence from vortexed
capsules versus that from sonicated capsules. Overall, however, the supernatant spectra
exhibit extremely low levels of fluorescence with respect to the capsules; for these

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Wavelength (nm)Fig. 7. Fluorescence spectra of capsules (thin solid) and supernatant subjected to mechanical insult(post-sonication: triangle, post-vortexation supernatant: diamond) and pH changes (pH 9: plus-sign,
pH 4: bold).

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experiments, a maximum of about 6% of fluorescence comes from dye free in supernatantrelative to capsules. These results indicate that, in qualitative terms, the polyelectrolyte
shells are stable under mechanical stress.
The capsules were also suspended in varying pH, and Fig. 7 contains supernatant
spectra collected from shells immersed in acidic and basic solutions. In comparison withthe mechanical tests, the changes in pH resulted in a much higher level of fluorescence inthe supernatant. In particular, shells suspended in pH 4.11 showed greater leaching ofdyes, showing approximately 15% when compared with capsule fluorescence. This result
was expected, as others have observed similar behavior.<2 1 l A possible explanation of thisoccurrence is that at high or low pH, the polyelectrolytes in the shell wall rearrange,
causing the pores on the shells to expand, and enabling diffusion of dyes out of thecapsules. Fortunately, in most potential physiological applications, the particles should bein a suitable pH environment. This may, however, be a limiting factor for other potential
uses of the capsules.

3.4 Sodium response tests

The response of loaded capsules to increasing sodium levels was studied as an initial
feasibility test. Figure 8 shows three representative spectra for different sodium concentra­
tions after normalization (418 nm) to correct for source drift. The sodium concentration
was increased in 10 mM increments, and it can be easily seen that the SBFI peak intensity
increases significantly with increasing sodium, though the plot only includes spectra for 0,
50, and 100 mM. These results are very promising, as the data indicate that the encapsu­
lated analyte-sensitive dye retains its indicator functionality.
Another critical point is the necessity of including an internal reference when making
fluorescence intensity-based measurements. Although it is not obvious from the normal-

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Wavelength (nm)Fig. 8. Representative fluorescence spectra (normalized to 418 nm) of capsules suspended in buffer
of increasing sodium levels. Spectra for concentrations of 0 mM (thick dashed), 50 mM (thin solid),
and 100 mM (thick solid) are shown.

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ized plots, the importance of the reference dye is clear when observing raw spectra. Due tovariations in the intensity of the laser source over the integration period, even replicate
spectra collected from the same sample vary significantly in overall intensity. Thus,
without a reference point, quantitative measurements would be extremely difficult because
any instrumental variations would lead to drift. For the system described here, replicate
scans overlap completely after normalization to the reference peak.
Figure 9 places the peak ratio changes with increasing sodium concentration on aquantitative scale. The first observation is that the peak ratios exhibit an approximately
linear relationship with increasing sodium concentrations. Secondly, this effect is ob­
served over the entire range of concentrations tested, and may extend beyond this point.
Another important point is that, according to the standard deviations described by error
bars, 20 mM accuracy can be achieved. The error bars decrease in size as the concentration
increases, most likely due to the development of a strong peak from SBFI from the lesswell-defined band superimposed on the strong MCA peak that is evident at low concentra­
tions. Thus, the response is clearly seen from the data, and it is realistic to believe that theaccuracy and range can be improved over that obtained for the simple example presented here.

4.

Conclusion

The paper describes a novel, extremely general technology for encapsulating fluores­
cence assays using nanoengineered liquid-core shells. The results of initial experiments
performed toward the understanding of loaded polyelectrolyte capsule properties are
extremely promising when considering potential sensor applications. It has been shownthat it is possible to encapsulate fluorescent probes in stable, polyelectrolyte microshells
with excellent controllability, and sodium-sensitivity expeliments suggest that encapsu-

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10

20

30

40

50

60

70

80

90

100

Sodium Concentration (mM)

Fig. 9. Fluorescence intensity ratio (493 nm: 418 nm) of capsule spectra with increasing sodium
concentration. Markers indicate the average peak ratio and error bars show one standard deviation;
all values were computed from three replicate scans at each concentration.

Sensors and Materials, Vol. 14, No. 6 (2002)

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lated fluorescent indicators can be useful as sensors. The long-term robustness of capsules
has been shown, and it is likely that these shells will prove flexible in the detection of otheranalytes.
The potential implications of the findings are that tremendous improvements may bemade over current methods being investigated for implantable biosensors. Because theinterior of capsules is similar to the ambient solution, they can accommodate a much larger
number of fluorophores per unit volume than any polymer-based approach. This results inincreased signal intensity as well as sensing range. Furthermore, the ability to control thewall thickness with a precision of a few nanometers hints at the possibility of tuning thepermeability of the capsules, thereby controlling the response time of encapsulated sen­sors. Transport will likely be much more rapid for capsules than for polymer carriers.
While these potential improvements are attractive, much work is needed to better grasp
the capabilities of capsules for use as biosensors. Future investigations will seek todetermine whether the above suggestions are accurate, and must consider a number ofbiosensor-related issues including calibration for quantitative measurements, reversibility,
biocompatibility, and interfaces with instrumentation. In addition, while the versatility of
the capsules as carriers for many fluorescent assays seems obvious, the limitations inloading different molecules must be identified. It is anticipated that future work will prove
capsules to be extremely general and useful in producing novel sensors for both intracellu­
lar and extracellular measurements.

Acknowledgment
This work is supported in part by grants from the Rockefeller Brothers Fund Charles E.
Culpeper Biomedical Pilot Initiative (01-153), the Louisiana Board of Regents ResearchCompetitiveness Subprogram (LEQSF(2001-04)-RD-A-18), and the National.Science
Foundation grant No. 0092001 "Micro/ Nanodevices and Systems". Any opinions,findings, and conclusions or recommendations expressed in this material are those of theauthors and do not necessary reflect the view of the National Science Foundation.

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