Double

Immobilized Sensing Gels for Op.cally Determining pH

Bradley Hambly Erno Lindner
(bhambly@memphis.edu) (elindner@memphis.edu)
Introduc)on
Tear lm composi.on is a crucial piece of evidence in the diagnosis

A.

of several diseases, e.g. Dry Eye Syndrome (DES). Pa.ents with DES and
infants have small volume of tear uid, i.e., it is dicult to collect a
testable volume of tear uid for analysis. The aim of this project is to
develop a microuidic pH sensor for the analysis of such samples. Op.cal

Materials & Methods

based vesicles as templates and tert-butyl methacrilate and butyl

methacrylate as monomers and ethylene glycol dimethacrylate as
crossing linking agent in the presence of a 500 micromolar solu.on of
Nile Blue A (NBA) dye. Three known concentra.on (mg NC/ g PVA) of
dye-loaded NC were mixed with 15% PVA (67,000 MW). [1%, 1.3%,
2%]
Thin-Film Fabrica)on and Op)cal Measurement: The NC:PVA
mixture was polymerized into 2 mm diameter thin-lms with varying
thickness using replica molding techniques. The thin-lm was placed
on the surface of a glass slide and covered with a
polydimethylsiloxane (PDMS) microuidic ow channel. A micro
spectrophotometer specically designed to hold the microuidic ow
cell assembly was used to measure the absorbance of the thin lm.
The thin-lms were analyzed for response .me (dA/dt = 0.005 or
dpH/dt = 0.06) and maximum absorbance for a 10 pH step change,
between pH 2.6 and 12.6, and calibra.on was performed on a 250
m 2% NC loading.

0.6
0.5

Absorbance

pH sensors have numerous advantages over tradi.onal pH sensi.ve

glass electrodes. However, the sensi.vity, sluggish response and short
life-.me make many pH optodes unsuitable for certain analy.cal
applica.ons. To overcome these drawbacks, high concentra.ons of
pH sensi.ve indicator dyes were encapsulated into ~200 nm diameter
nanocapsules (NC) with 1 nm thick porous walls. The capsules were
immobilized into porous polyvinyl alcohol (PVA) gels for a robust and
versa.le sensor plaRorm design1,2 (Fig. 1). The nanocapsule-loaded
gels were molded into thin-lms and used as sensing elements in a
microuidic ow cell. The absorbance and response .me of the
sensing gels with entrapped dye-loaded nanocapsules (double
immobiliza.on) were studied as a func.on of the sample pH using
tradi.onal UV-VIS spectrophotometry.





Figure 1. Illustra)on of dye-loaded porous NCs (leE) and sensing gels
with embedded nanocapsules (right).




Nanocapsule
Prepara)on: Porous NCs were prepared using surfactant

0.7

0.4
0.3
0.2

B.

0.1
0
5.5

6.5

7.5

pH

8.5

9.5

10.5
250 m

750 m

Figure 5. LeE: Calibra)on of a 2% NC 250 m PVA gel by ow injec)on analysis (~450 L loop).
Right: Reproducibility of individual of 1% NC loaded gels (n = 8 over two days)
Figure 2. A) A cross-sec)onal view of the microuidic ow cell optode with thin-lm sensing gel.
B) LeE: images of the MUTOH America, Inc. microuidic spectrophotometer unit. Light is
provided using an integrated Warm White LED. Flexible tubing connects to a peristal)c pump and
waste container. The unit connects to a laptop computer installed with the acquisi)on soEware
via USB. Right: Image of the microuidic ow cell with thin-lm sensing gel contained within the
channel.
450 L

300 L

50 mM NaOH (pH 2.6)

0.5 mL/min

85 L

S
Figure 3. Experimental setup for calibra)on by ow analysis. The sample is injected
into the loop and introduced into the ow system. Once the sample reached the
ow cell, the ow was stopped to allow for diusion into/out of the NC loaded gel.
A 2% NC loaded gel was used in this analysis with solu)ons ranging from pH 3 to 10.
Gel spectra in 50 mM NaOH was used as the reference spectra as well as the carrier
solu)on.

Results and Discussion

Figure 6. Flow-injec)on calibra)on of pH standards (3.00, 5.00, 7.04, 9.07, 4.05, 6.03, 8.15 and
10.06 respec)vely). Flow was stopped 45 seconds aEer injec)on and remained stopped for 3
minutes to allow for diusion into the sensing NC loaded gel.

Conclusions
pH optodes were fabricated using double immobilized Nile Blue A in a
porous NCs set in PVA . The pH dependence of the porous NC-based optodes
were measured in a sampling capillary and in a microuidic ow cell devices.
The performance characteris.cs of the NC pH optodes were evaluated using
the Mutoh spectrophotometer. The 250 m sensing gel-based optode had
excellent reproducibility and symmetrical response .me (p = 0.5) of 324
seconds.
pH response was determined to have a sensi.vity of 0.86 absorbance/pH,
and a ~0.003 pH error is expected in the measurement.
Future work to include reduc.on of the variability of the measured
absorbance, reduc.on of the sample requirement for measurement (sub
100L goal), and increasing sensor sensi.vity by further increasing NC
concentra.on in the sensing gels.

Figure 4. An absorbance vs. )me curve for a 250 m thick sensing gel loaded with 1% NC in
response to a 10 unit pH step change (12.6 2.6). Average absorbance from experiments
performed on three dierent days was determined to be 0.150(0.006) with a reproducibility of
3.7%.

References

[1] Dergunov SA, Miksa B, Ganus B, Lindner E, Pinkhassik E. Nanocapsules with "invisible" walls. Chem Commun (Camb). 2010 Mar 7;46(9):1485-7.
PubMed PMID: 20162156
[2] Kim MD, Dergunov SA, Lindner E, Pinkhassik E. Dye-Loaded Porous Nanocapsules Immobilized in a Permeable Polyvinyl Alcohol Matrix: A
Versa.le Op.cal Sensor PlaRorm. Analy.cal Chemistry2012 84 (6), 2695-2701