Fluorescence-based sensing systems offer potential for noninvasive monitoring with implantable devices, but require carrier technologies that provide suitable immobilization, accessibility, and biocompatibility. Recent developments towards this goal include a competitive binding assay for glucose that has been encapsulated in semipermeable microcapsule carriers. This paper describes an extension of this work to increase the applicability to in vivo monitoring, wherein two significant developments are described: (1) a near-infrared resonance energy transfer system for transducing glucose concentration, and (2) novel hybrid organic-inorganic crosslinked microcapsules as carriers. The quenching-based assay is a competitive binding (CB) system based on apo-glucose oxidase (AG) as the receptor and dextran as the competitive ligand. The encapsulated quencher-labeled dextran and near infrared donor-labeled glucose receptor showed a stable and reversible response with tunable sensitivity of 1–5%/mM over the physiological range, making these transducers attractive for continuous monitoring for biomedical applications.
It is well known
that frequent monitoring of glucose concentrations and appropriate
countermeasures can help in achieving euglycemia and minimizing secondary
complications of diabetes [
While several approaches
for fluorescence-based glucose sensing have been developed using Concanavalin A [
We also
developed a carrier system that provides accessibility to the analyte
while preventing movement of the molecules outside the region of intended use [
While these
previous reports represent necessary steps toward a viable monitoring system, a
number of additional obstacles to clinical acceptance remain. Three improvements that would help move
toward the ultimate goal are an increase in the operating wavelengths, a more
facile capsule fabrication and encapsulation process, and a stable capsule
formulation. Longer wavelengths will enhance
penetration of light through tissue. However, while a large selection of
organic dyes and inorganic materials have been identified and used in RET
systems [
These problems
were solved in this work by (1) converting the transduction scheme from a
system in which the acceptor emits photons to one in which the acceptor is
deexcited through nonradiative pathways, where a near-infrared dye (Alexa Fluor
647) is attached to the glucose receptor and a broadband quencher (QSY21) is
attached to the competitive ligand, and (2) encapsulating the assay in hybrid
polymer-silica microshells. A number of
different microcapsules and construction strategies with controllable shell
thickness and properties have been proposed based on the versatile LbL
self-assembly process [
This paper describes the investigation of an NIR glucose sensor comprised of competitive binding assay encapsulated in hybrid microcapsules, and compares the performance characteristics with those obtained for the FITC/TRITC and TRITC/Cy5 energy transfer pairs. We also report a novel method for the encapsulation of macromolecules into silane-modified PSS/PAH microcapsules that eventually form organo/inorgano hybrid microcapsules. The hollow silane-based microcapsules slowly form an interpenetrating silica network due to the hydrolysis and subsequent condensation of silane. While the thorough characterization of the hybrid microcapsules will be described in a separate report, the details of the materials and methods used to fabricate these unique structures are provided here for completeness.
A schematic of a
glucose sensor based on this quenching mechanism is shown in Figure
Schematic of the RET quenching system for glucose monitoring based on the competition between dextran and glucose for binding sites on apo-GOx.
The homogeneous
energy transfer assay described above can be mathematically described by
expressions for the competitive binding reactions between the immobilized
receptor (
A key aspect of
this system is the dependence of the response on different concentrations of
assay components, in both absolute and relative senses. Using the equations above,
the graphs in Figure
The response of the sensing system can be
characterized by the effective dissociation constant of the sensing assay, also
referred to as the concentration at which half of the full response is observed
Effect of dimensionless analyte
concentration on relative response
Estimated assay response to analyte, in terms
of percentage increase in unquenched receptor.
Inset gives value for multiplier used in set the concentration of assay
(relative to
Glycidyl 3-(trimethoxysilyl)propyl ether
(glycidyl-silane), GOx (G-2133), sodium
poly(styrene sulfonate) (PSS, MW ~1 MDa), poly(allylamine hydrochloride) (PAH,
MW 70 kDa),
A UV-Vis spectrometer
(Perkin Elmer Lambda 45) was used to collect absorbance spectra. The slit size
(4 nm) and scanning speed (480 nm/min) were held constant throughout all the
experiments. A scanning fluorescence spectrometer (QM1, Photon Technology
International) with an extended-wavelength PMT (R928) was used to collect
fluorescence emission spectra while exciting the sample at 640 nm. Confocal
images were taken with a Leica TCS SP2 microscope equipped with a 63X oil
immersion objective and a red He-Ne excitation laser. Counts and sizes of
microcapsules were obtained with a Beckman Coulter counter (Z2) using a 100
The glucose assay concentration was optimized by performing a simple titration experiment to obtain maximum change in signal for a given analyte concentration. The concentrations of the sensing assay elements were optimized based on the concentration-dependent quenching of AF647 by QSY21. To observe the quenching behavior and confirm RET, aliquots of 700 nM QSY21-dextran (QSY-dex) solution were titrated into 17.5, 35, and 70 nM AF647-AG (AF-AG) in a stepwise manner, followed by measurements of fluorescence emission.
The apo-GOx and
amino-dextran used in all experiments were labeled with AF647 and QSY21 at ratios
of 3.2 and 2.4, respectively. The quenching process between AF-AG and QSY-dex
was monitored using the fluorescence spectrometer by exciting the sample at 640 nm and collecting the emission across the range of 650–750 nm. Initially, ~16.5 picomoles of AF647-AG were added to 0.4 mL of DI water. Based on quenching studies,
the initial concentration of QSY-dex for 41 nM AF-AG was selected to be 143 nM. To assess the relative
affinity of apo-GOx for glucose and QSY-dex, recovery in the AF647 emission was
observed during the stepwise addition of aliquots of 500 mM
The PSS and PAH solutions used for
building multilayer films were prepared in DI water at 50 mM (concentration based
on monomer). For LbL assembly of PSS/PAH-silane, 20
Process for organic/inorganic hybrid microcapsule fabrication and
loading (a)–(c); encapsulation of assay reagents into
hollow microcapsules (d)–(f); and (g) surface
potential values obtained after coating each layer on
Microcapsules
with the
The AF750-labeled microcapsules loaded with AF-AG and QSY-dex were
suspended in DI water, and an initial fluorescence spectrum was collected. Glucose
solution (500 mM) was then titrated into the microcapsule suspension, as was performed for the solution-phase studies.
The change in the relative emission intensities at 664 and 780 nm was calculated
for each spectrum and plotted with respect to glucose concentration. To confirm
theoretical prediction on the effect of receptor/competitive ligand
concentration and to evaluate the possibility of tailoring the sensor response,
these experiments were repeated with four different concentrations of capsules,
(
To test the reversibility of the
microcapsule sensors, exposure to glucose in random order with respect to
concentration was performed. To achieve
this, microcapsules were dispersed in DI water (
AF-AG samples
exhibited decreasing emission intensity during the titration of QSY-dex,
following a pattern predicted by equilibrium binding (Figure
Quenching of AF647-AG in response to QSY21-dextran titration.
Taking the optimum assay concentrations obtained from the quenching experiments
into consideration, an assay mixture containing 142 nM QSY-dex and 42 nM AF-AG was
prepared in DI water and subjected to sequential glucose additions. As hypothesized, the added glucose displaced
dextran from apo-GOx, resulting in decreased quenching and correspondingly
enhanced fluorescence emission at 664 nm (Figure
Normalized fluorescence spectra (a) and percentage change in AF647 emission with the addition of glucose into AF-AG/QSY-dex complexes (b). Lines are regression curves used to identify trends. Error bars indicate one standard deviation of three replicate measurements. The inset of (b) indicates the QSY21-dex concentrations.
The surface
potential of microparticles alternated with the addition of each layer, (Figure
The intensity of fluorescence at 664 nm
increased with each addition of
We note that this
behavior is consistent with previous studies with visible dyes [
Given that physiological
sensing is the goal, and that pathophysiological glucose levels experienced by
diabetics may range from over 0–30 mM (0–600 mg/dL),
matching the sensor response to this range requires proper selection of reagent
concentration. As can be seen in Figure
Loading concentration and response properties for samples with varying capsule concentration.
Capsules/mL | QSY-dex (picomol) | AF-AG (picomol) | Sensitivity ( | Linear range (mM) | |
---|---|---|---|---|---|
18 | 23 | 5.15 | 0–10 | 4.1 | |
37 | 46 | 3.09 | 0–30 | 10 | |
73 | 92 | 1.97 | 0–40 | 20 | |
184 | 146 | 0.8 | 0–80 | 60 |
Fluorescence spectra of the microcapsules loaded with sensing assay and reference dye (AF750) with the addition of glucose solution. (a) All the spectra are normalized to the reference peak at 776 nm. (b) Relative change in AF647 : AF750 peak ratio with the addition of glucose solution. Error bars indicate one standard deviation of three replicate measurements.
Exposing
the microcapsules (
Percentage change in AF647:AF750 peak ratio with the addition of random
An
efficient RET quenching approach was developed as the transduction mechanism for
a dextran/apo-GOx competitive binding assay, extending the operating region
into the near-infrared. The sensing assay elements (a quencher-labeled dextran,
fluorophore-tagged apo-glucose oxidase, and a fluorescent reference dye) were
entrapped in microcapsules using a facile and efficient silane-based
encapsulation procedure. Microcapsules containing labeled dextran/apo-GOx
complexes showed glucose sensitivity of ~1–5%/mM, which is comparable to the
original assay operating in the visible region and is among the most sensitive ever reported. Thus, this system is superior to previous
iterations, as it possesses the following desirable qualities: (1) the receptor
is nontoxic [
The authors gratefully acknowledge the National Science Foundation (NIRT Grant no. 0210298) and NIH (R01EB000739). Any opinions, findings, and conclusions/recommendations expressed in this material are those of the authors and do not necessarily reflect the view of the National Science Foundation.