Crystalline Colloidal Array
Chemical Sensing Devices
Sanford A. Asher
Dept. of Chemistry
University of Pittsburgh
Pittsburgh, PA 15260
412-624-8570
asher@pitt.edu
Sanford A. Asher, Department of Chemistry
Mesoscopically Periodic Materials
CCA
Self-Assembling
Diffracting Structure
Fragile
Optical Filters
PCCA
Hydrogel Volume
Phase Transition
Robust
Tunable Spacings
Optical Filters
SCCA
IPCCA
Rigid 3-D Periodic
Materials
Rugged
Optical Filters
NLO Switches
Optical Limiters
Electronically
Chemically
Thermally
Responsive
Materials
Smart Materials
Agile Optical
Filters
Chemical Sensing
Display Devices
PHOTONIC CRYSTALS
Sanford A. Asher, Department of Chemistry
CRYSTALLINE COLLOIDAL SELF-ASSEMBLY:
MOTIF
FOR
CREATING SUBMICRON
PERIODIC SMART MATERIALS
Sanford A. Asher, Department of Chemistry
Outline
ƒ CCA and PCCA Photonic Crystal
Fabrication
ƒ Hydrogel Volume Phase Transitions
ƒ Photonic Crystal Chemical Sensors
-Donnan Potential Pb2+ Sensor
-Glucose Oxidase Glucose Sensor
-Metal Cation Sensors
-Glucose Sensors
-Creatinine Sensors
-Antibody sensors
Crystalline Colloidal Arrays Self-Assembly
z fabricated from monodisperse, highly charged colloidal particles
Dialysis /
Ion Exchange
Resin
- + ++ - + +- - -+ +
- ++-- +-+
+ -+
+
+
- -- - +++ +
- - + - +
+
+
Self-assembly
FCC
~ 1013 spheres/cm3
-+- - +
+
+
+--+
+ - - +
+- +
+
+
-
- - +
-- --+
- +
++ +-
-- - +
-+ - - +
-
+
++ -+
- - - + - - - -+
- - -- - + - +
+
- - +- -- +
- - ---+ - - +
-- -+ -- +
+ -- - -+ +
- +- +
- ++
-+ +
+
+
+
++ -- - -+
-+
+ + -+ - -- - +
- - -- - - + - - ++
- - +-- - +
++ - - +
-+
+-
-- -- +
+ - - +
+-
- - - -+
-- + +-+ -+
-- --- + +
-- -+
+
- - +- +
+ -+
- -+ - +
-+- - +
Crystalline Colloidal Array
z spacing dependent only upon
particle number density and
crystalline structure
Holtz, Asher et al J. Am. Chem. Soc. 1994, 116, 4497
What Drives CCA Self-Assembly?
+
+
SO3- H H -O3S
H+ -O S
3
H+
-O
SO3-+
3S
-O
H+
H
+
SO3- H
U
SO3- +H
3S
r
2
κa
2
⎤ e − κr
Z e ⎡ e
U (r ) =
ε ⎢⎣ 1 + κ a ⎥⎦ r
2
2a
Negatively charged particle
Sphere Radius
Medium Dielectric Constant
Interaction Potential Energy
2
4
π
e
(n p Z + ni )
κ2 =
ε kB T
Ionic Impurities
Particle concentration Shear boundary
Debye layer thickness
1
κ
(in pure water ) ~ 700 nm
r
Sanford A. Asher, Department of Chemistry
+
Bragg Diffraction
λ0
+
+
+
+
- - - - - ------- - - +
+
+
- - - - - ------ - - -+
θB
+
- - --------- - - --
+
d ~ 200 nm
+
+
- - - - - ------- - -+
+
- - - - - ------- - +
- - - - - -----+ - - - --
+
d
-- - - - ----- +
---- - -
+
(111) FCC CCA
- - - - - ---------- - -+
+
+
+
+
+
+
- - - - - -------- - -- +
+
- - - - - ------- - - -+
-- - - - ------- - +
+
- - - - - -------- - - -- +
+
- - - - - ------ -+ --
mλo = 2ndsin θ
+
λ0 = wavelength of diffracted light
n = refractive index of system
d = interplanar spacing in crystal
θB = Bragg glancing angle
Diffracted Intensity, ID
+
+
+
- - - - - --------- - --
500
600
700
Wavelength / nm
800
All Light Diffracted-Finite Widths-Top Hat Profiles
PCCA Fabrication
+
- - +- - -+ - - +
+---- - - --- - ++
+
+- - --- - +- - - + +- - --- - +-- - - - -+
-- - ++ - - -- +
-+ -+ - --- - +-- -+
+
- - -- - - - -- + - -- - - -- +
-- --- +
+
+
+
- -- - + - - +- -+ -+--+
+
+
- -- +- - +- - -- - - - -+- -- -- - -- - ++
- - -- +
+
- -+- --- - --+ -- -- - -- - --+
-+ - - + +- - - --+ - -- - -+
+ --- --- -+
+
+
+
-+ - - +
- - +- - - - --- - - - --- - +- - - -+
+
+- --- - +- - - + +- - --- - - +- - - -+
-- - ++ - - -- +
- + --- -+- - - + - -- - ++
- - -- - - - -- + - -- - - -- +
-- --- +
+
+
+
- -- - + - -- -+
+- -+ +-+ - -- - +
- -- - ++- - -- - -- -- - - - -+- - - +- - -- +
+
+- -+- --- - --+ -- -- -- --- +- - -- - -+ - - + +- - - --+ - - -+
+ --- -+- - - +-
Acrylamide
Bisacrylamide
Photopolymerize
+
Self Assembly Motif for Creating Submicron Periodic Materials. Polymerized
Crystalline Colloidal Arrays,
S. A. Asher, J. Holtz, L. Liu, and Z. Wu, J. Am. Chem. Soc. 116, 4997-4998 (1994).
+
Diffraction from CCA and PCCA
CCA
PCCA
Hydrogels are Responsive
Enabling Materials
FREE ENERGY CONTRIBUTIONS TO GEL VOLUME
Δ Gtot = Δ Gmix + Δ Gion + Δ Gelas
+
Δ Gion
Δ Gmix
+
Δ Gmix
Δ Gelas
Δ Gelas
Sanford A. Asher, Department of Chemistry
Classical Model
*
• For Nonionic Gels
ƒ Free Energy:
ΔGtot = ΔGmix + ΔGelastic
ΔGmix = k BT [n ln(1 − φ ) + nχφ ]
ΔGelastic
From Stress vs. Strain
3k BT ⎛ ν e ⎞ ⎡⎛ V ⎞
⎜⎜ ⎟⎟ ⎢⎜⎜ ⎟⎟
=
2 ⎝ Vm ⎠ ⎢⎝ Vm ⎠
⎣
2/3
⎤
− 1⎥
⎥⎦
ƒ Osmotic Pressure: Π = - (μH2O - μ0H2O) / VH2O
⎛ V2 ⎞ ⎛ λ2 ⎞
Using ⎜⎜ ⎟⎟ = ⎜⎜ ⎟⎟
⎝ V1 ⎠ ⎝ λ1 ⎠
Πtot = Π mix + Πelastic
Π mix
3
3
6
RT ⎡ ⎡ ⎛ λ0 ⎞ ⎤ ⎛ λ0 ⎞
⎛ λ0 ⎞ ⎤
=−
⎢ln ⎢1 − ⎜ ⎟ ⎥ + ⎜ ⎟ + χ ⎜ ⎟ ⎥
VH 2O ⎢ ⎢⎣ ⎝ λ ⎠ ⎥⎦ ⎝ λ ⎠
⎝ λ ⎠ ⎥⎦
⎣
At Equilibrium Πtot = 0
Π elastic = −
3
RT ⎛ ν e ⎞⎛ λm ⎞
⎜⎜ ⎟⎟⎜
⎟
2 ⎝ Vm ⎠⎝ λ ⎠
χ ≈ 0.5
*Flory,
“Principles of Polymer Chemistry” (1953)
Free Energy Contributions to Gel Volume
ΠM
2
⎡
RT
⎛ Vo ⎞ Vo
⎛ Vo ⎞ ⎤
=−
⎢ln⎜1 − ⎟ + + χ ⎜ ⎟ ⎥
Vs ⎢⎣ ⎝ V ⎠ V
⎝ V ⎠ ⎥⎦
ΔΔG
Gmix
mix
acetone
water
Gmix
ΔΔG
mix
Free Energy Contributions to Gel Volume
(
Π I = RT c+ + c− − c − c
*
+
*
−
)
+
+
+
+
Δ Gion
+
+
analyte
salt solution
solution
+
+
+
+
+
+
+
Free Energy Contributions to Gel Volume
1
⎡
⎤
3
RT ⋅ ncr ⎢⎛ Vm ⎞ 1 Vm ⎥
ΠE = −
⎜ ⎟ +
Vm N av ⎢⎝ V ⎠ 2 V ⎥
⎣
⎦
molecular recognition agent
analyte
analyte
water
solution
Δ G elas
Sanford A. Asher, Department of Chemistry
Poly(N-isopropylacrylamide) : T Dependent ΔGMIX
Poly(N-isopropylacrylamide)
(PNIPAM) undergoes a
reversible phase transition
when heated above 32.1 oC.
This coil-globule transition is
analogous to a liquid-vapor
phase transition. The recipe
and synthesis conditions
determine the extent of volume
changes and whether they are
continuous or discontinuous.
Chemical Structure of NIPAM
N
O
Sanford A. Asher, Department of Chemistry
Sanford A. Asher, Department of Chemistry
Sanford A. Asher, Department of Chemistry
Outline
• CCA and PCCA Photonic Crystal
Fabrication
• Hydrogel Volume Phase Transitions
• Photonic Crystal Chemical Sensors
-Donnan Potential pH and Pb2+ Sensor
-Glucose Oxidase Glucose Sensor
-Metal Cation Sensors
-Glucose Sensors
-Creatinine Sensors
-Antibody sensors
Hydrolysis of PCCA
NaOH
Nonionic
Ionic !
ƒ For Ionic Gels …
VPCCA = f (pH & Ionic Strength)*
ƒ Can monitor VPCCA from Diffraction Wavelength
*Tanaka,
Sci. Am. (1981)
Flory’s Model II
• Ionic Gels
Πtot = Π mix + Πelastic + Πion
From ion concentration imbalance inside and outside gels
K a [COOH ]
Π ion
*
*
[
]
2
(
)
= i COOH − Cs − Cs =
−
2
C
sη
+
RT
Ka + [H ]
extent of ionization
Ionization
• At Equilibrium: Πtot = 0
Πion = −(Π mix + Π elastic )
Δ [electrolyte]
Effect of pH on VPCCA
• Low pH
• High pH
maximum volume occurs at pH 9.6
Effect of Ionic Strength on VPCCA
Diffraction wavelength decreases as ionic strength increases
pH DEPENDENCE OF DIFFRACTION
• Near pH ≈ 7
Negligible
• Z-1 vs. [H+]
Π ion K a [COOH ]
*
=
−
2
(
C
s − Cs )
+
RT
Ka + [H ]
K a [COOH ]
1
(
)
−
Π mix + Π elastic = Z =
RT
Ka + [H + ]
Taking inverse of both sides:
Z
−1
[H + ]
1
=
+
K a [COOH ] [COOH ]
Plotting Z-1 vs. [H+] gives:
Slope =
1
K a [COOH ]
1
Intercept =
[COOH ]
Ka = 7.7×10-6 M
[COOH] = 9.2×10-4 M
≈ 0.1% Hydrolysis !
Modeling pH and Ionic Strength
Response
• pH
• Ionic Strength
Can Now Model PCCA Volume Changes !
Sanford A. Asher, Department of Chemistry
Chelation of the Pb 2+ ion results in immobilization of the
counterion w hich results in an osmotic pressure which
swells the gel and red shifts the diffraction in proportion to
the analyte concentration.
Pb 2+ Pb 2+
Pb 2+
Pb 2+
Extinction
Pb 2+
Pb 2+
Pb 2+ Pb 2+
41ppb
2.0
410 ppb
water
4.1 ppm
1.5
1.0
0.5
0
Diffraction Wavelength Shift / nm
400 450 500 550 600 650
Wavelength / nm
160
140
120
100
80
60
40
20
0
H
H
N
O
O
O
O
C
N
O
C
C
O
H
C
C
N H
N H
H
H
N H
H
C
N
H
H
O
O
H
O
O
H
N
O
O
H
H
O
O
C
O
C
C
O
N H
H
O
O
2
20
200
O
Pb 2+O
O
0.2
H
C
C
N H
N H
H
0.02
N
N H
O
C
O
O
2000
Lead Concentration / ppm
Intelligent Polymerized Crystalline Colloidal Arrays: Novel Chemical Sensor Materials, J. H. Holtz,
J. S. W. Holtz, C. H. Munro, and S. A. Asher, Anal. Chem. 70, 780-791 (1998).
Sanford A. Asher, Department of Chemistry
Sour ce
M onochromator
De te ctor
Fibe r Couple rs
Bifur cate d Fiber
% Reflectance
No
Pb2+
Pb 2+
PCCA sens or tip
20
8 m M Pb 2+
10
0
400
450
500
550
600
Wavelength / n m
650
700
Sanford A. Asher, Department of Chemistry
Glucose Oxidase Reaction Mechanism
gluconic acid
glucose
GOx
GOx
GOx
+
+
O
R
O
O2
GOx
GOx
R
O
R
R
H3C
N
H3C
N
O
+ glucose
H3C
N
H3C
N
N
H
O
H
Oxidized Flavin
O2
-
O
N
N
N
H
O
Reduced Flavin
R
H3C
N
H3C
N
H
N
O
N
H
O
Oxidized Flavin
+
H2 O 2
Sanford A. Asher, Department of Chemistry
GOx
GOx
0.1 mM
1.2
Extinction
0.2 mM
0.3 mM
0.4 mM
water
1.0
0.5 mM
GO x
0.8
glucose
0.6
gluconic
acid
0.4
0.2
450
500
550
600
650
Wavelength / nm
700
750
Sanford A. Asher, Department of Chemistry
Outline
• CCA and PCCA Photonic Crystal
Fabrication
• Hydrogel Volume Phase Transitions
• Photonic Crystal Chemical Sensors
-Donnan Potential pH, Glucose and Pb2+
Sensor
-Metal Cation Sensors
-Glucose Sensors
-Creatinine Sensors
-Antibody sensors
Preparation of metal cation sensor PCCACS
O
O
PCCA
0.1 N NaOH
NH2
10 % TEMED
Polyacrylamide
1.5 hrs
PCCA
PCCA
OH
hydrolyzed
PCCA
NH2
EDC
Photo-polymerization
using UV light
1.5 hrs
1.5 hrs
N
OH
8-hydroxyquinoline
O
O
O
NH2
Acrylamide
+
O
N
N
H
H
bisacrylamide
+ CCA (125 nm, 8 wt.%)
PCCA
NH
N
OH
8-hydroxyquinoline
PCCACS
Other 8-hydroxyquinoline based metal ion sensors:
Bronson et. al. J. Org. Chem. 2001 66(14), 4752-4758.
Response of PCCACS to aqueous Cu2+ solutions
54 μM
Diffraction intensity / a.u.
1 μM
0.5 mM
0.54 μM
11 nM No Cu2+
54 μ M
0.54 μ M
1 μM
500
5 mM
0.5 m M
550
2 mM 5 mM
11 nM
buffered
saline
2 mM
600
650
700
λ / nm
750
800
After washing, Cu2+ treated PCCACS stays blue shifted, indicating permanent sequestering
of Cu2+ in the hydrogel.
Proposed mechanism of Cu2+ sensing with PCCACS
O
O
NH
NH
O
N
NH
O
N
2+
N
OH
Metal ion sensor
PCCACS
Cu
aqueous
medium
low metal ion
concentration
O
Cu
2+
O
+ Cu
N
2+
- Cu
2+
2+
Cu
L
L
L L
Cu
2+
O
N
HN
O
Cu(hydroxyquinolate)2
complex formation
log(Kf) = 21.87
Collapse of hydrogel polymer
network volume due to formation
of additional crosslinks
Diffraction maximum
blue shifts
HN
O
Cu(hydroxyquinolate)
complex formation
log(Kf) = 10.70
Expansion of hydrogel polymer
network volume due to breaking
of crosslinks
Diffraction maximum
red shifts
Theoretical fit for the experimental Cu2+ response
Cu
2+
s to ic h io m e try
0 .0 1 0 .1
1
Diffraction maximum / nm
750
700
650
600
550
10
-7
-5
-3
10
10
2+
C u conc. / M
0 .1
Hence, we can obtain a good fit for the experimental diffraction maxima for various Cu2+ concentrations.
The lower concentrations cannot be fit well, due to the very high formation constant for the 2:1 complex
sites. PCCACS can be used as a dosimeter at sub-stoichiometric Cu2+ concentrations, and as a
reversible sensor for higher concentrations.
Sanford A. Asher, Department of Chemistry
Outline
• CCA and PCCA Photonic Crystal
Fabrication
• Hydrogel Volume Phase Transitions
• Photonic Crystal Chemical Sensors
-Donnan Potential pH, Glucose and Pb2+
Sensor
-Metal Cation Sensors
-Glucose Sensors
-Creatinine Sensors
-Antibody sensors
First Generation
Of Photonic Crystal
Glucose Sensors
Sanford A. Asher, Vladimir L. Alexeev, Alexander V. Goponenko,
Anjal C. Sharma, Igor K. Lednev, Craig S. Wilcox, David N. Finegold
Photonic Crystal Carbohydrate Sensors: Low Ionic Strength Sugar
Sensing. JACS, 2003 ASAP Web Release Date: 22-Feb-2003
Chemical Modification
of Acrylamide Backbone
OH
PCCA
NH2
O
AA PCCA
NaOH
TEMED
B
PCCA
OH EDC
O
OH
B
OH
OH
NH
PCCA
O
NH2
AA-BA Sensor
Equilibria Associated with 3- Acetamidophenylboronic Acid - Glucose Binding
O
O
OH
HN
B
+ OH , K1
OH
OH
-B
HN
HN
OH
B
O
O
OH
+ Glu, K4
+ Glu, K3
O
OH
+ OH , K2
O
OH
OH
O
HN
OH
OH
-B
O
O
O
OH
OH
Glucose concentration in mM
0.2 0.4
0
0.6
500
600
1
2 10 20
700
Wavelength / nm
Diffraction maximum / nm
Diffraction Intensity / a.u.
Glucose Concentration Dependence of the Diffraction
of the Polyacrylamide-Boronic Acid PCCA at pH 8.5
750
700
650
600
550
500
0
5
10
15
20
Glucose concentration / mM
Sugar Concentration Dependence
of the Hydrogel Swelling Degree
1.5
1.4
1.4
1.3
1.2
D-glucose
D-galactose
1.1
1.0
0
40
80
120
Sugar concentration/mM
λ/λ0
λ/λ0
1.3
D-fructose
D-mannose
Methylglucose
1.2
1.1
1.0
0
40
80
120
Sugar concentration/mM
Second Generation
Of Photonic Crystal
Glucose Sensors
Vladimir L. Alexeev, Anjal C. Sharma, Alexander V. Goponenko,
Sasmita Das, Igor K. Lednev, Craig S. Wilcox, David Finegold
and Sanford A. Asher. Photonic Crystal Carbohydrate Sensors:
High Ionic Strength Glucose Sensing. Anal. Chem. in press
Development of Cross Linking Motif for
Glucose Sensing of Bodily Fluid
Develop Recognition Motif Where
Glucose Forms Crosslinks Upon
Binding
OH
O
O
HO
O
B
-
O
O BHO
R
R
Solution:
O
O
OH
OH
O
-B
PCCA
HO
O
O
O
O
O
O
+Na
O
O
+
O
GLU
Na
O
O
-
B
OH
O
PCCA
BA-AA-PEG Sensor Diffraction Dependence on
Glucose Concentration
Diffraction intensity/a.u.
G lu c o s e c o n c e n t r a t io n in m M
10 20 1
525
550
0
2 mM tris-HCl, pH 8.5;
150 mM NaCl
575
600
W a v e le n g t h / n m
625
Specificity of the BA-AA-PEG
to Different Sugars
CH2OH
O
H
H
H
OH
HO
H
H, O H
CH2 OH
O
H
H
H
H
HO
OH
OH
D-Glucopyranose
CH2 OH
O
HO
H
H H, OH
OH
H
OH
H
H, OH
OH
D-Allopyranose
H
H
HO
H
H
O
OH
OH
CH OH
2
OH
H
O
HOH 2C
H
H, OH
H
H
OH
OH
D-Ribose
CH2OH
O
H
H
OH OH H, OH
HO
H
H
β-D-Fructopyranose D-M annopyranose
Bis-Bidentate Complex Formation Between
One Glucose and
Two Boronates
6
H
5
HO O 4
O
-B HO 3
H H
H
O
1
2
H
O O
-B
OH
AA chain
AA chain
Pyranose form
A Bis-Bidentate Complex Between Glucose and
Two Boronates Stabilized by PEG and Sodium
O
OH
OH
O
-B
HO
O
O
O
+
Na
O
O
O
O
+
O
O
O
Na
-
B
OH
OH
Third Generation
Of Photonic Crystal
Glucose Sensors
4-amino-3-fluorophenylboronic acid
pKa = 7.8
OH
F
H2N
B
OH
Artificial tear fluid
The tear fluid composition was taken from
the Geigy Scientific Tables, v.1, p.181-184, 1981
Sodium bicarbonate:
26 mM
Chloride (K+ + Na+ ):
124 mM
Calcium (chloride+carbonate): 0.57 mM
Urea:
5 mM
Ammonia:
3 mM
Vitamin C:
0.14 mg/100 ml
Citric acid:
0.6 mg/100 ml
Lactate:
2.5 mM
Pyruvate:
0.2 mM
Total albumin:
3.94 g/L
Total globulin:
2.75 g/L
Lysozyme:
1.7 g/L
Buffered with 2 mM tris-HCl, pH 7.4
Response to glucose in
artificial tear fluid
a rtific ia l te a r flu id
b u ffe re d s a lin e a t p H 7 .4
Diffraction blue shift/nm
Diffraction blue shift/nm
0
50
100
2 ru n s in a rtific ia l
te a r flu id
0
50
100
150
150
0
5
10
G lu c o s e /m M
15
0
5
10
G lu c o s e /m M
15
For high ionic strength solutions, the Donnan
potential is negligible and ΠΜ + ΠΕ = 0
total number of crosslinked chains is
Vol relaxed hydrogel
ncr = ncro + 2 nB2G
Flory-Huggins interaction parameter
From K
1/ 3
⎡
RT ⋅ n ⎛ Vm ⎞
∂ΔGE
1 Vm ⎤
ΠE = −
=−
⎢⎜ ⎟ −
⎥ − RT ⋅ cB2G
∂V
Vm ⎢⎣⎝ V ⎠
2 V ⎥⎦
0
cr
2
⎡
∂ΔGM
RT ⎛ V0 ⎞ V0
⎛ V0 ⎞ ⎤
=−
ΠM = −
⎢ln⎜1 − ⎟ + + χ ⎜ ⎟ ⎥
∂V
Vs ⎢⎣ ⎝ V ⎠ V
⎝ V ⎠ ⎥⎦
V is the secret
unknown ~ λ3
Molar vol Vol hydrogel
water
Flory-Huggins
parameter
Vol dry
hydrogel
Conclusion
1. Photonic
Glucose Sensor senses glucose
in bodily fluids at physiologica glucose
concentrations
2. We are developing it for continuous invivo glucose monitoring in tear fluid
3. A start-up company Glucose Sensing
Technologies, LLC is commercializing our
glucose sensor
A General Photonic Crystal Sensing
Motif: Creatinine in Bodily Fluids
Anjal C. Sharma, Tushar Jana, Rasu Kesavamoorthy, Lianjun
Shi, Mohamed A. Virji, David N. Finegold
and Sanford A. Asher*
General Clinical Sensing Motif
creatinine diffuses into hydrogel and
binds to enzyme
=
creatinine
washing
creatinine hydrolysis
produces OH-
enzyme
titrant
-
OH- deprotonates titrant
Hydrogel
swells
causing red-shift
-
OH
-
40
35
30
25
20
15
10
5
0
0.3 mM 0.5 mM
0.7 mM
0 mM 0.1 mM
1 mM
Diffraction Intensity / a.u.
Diffraction red shift / nm
Response to Creatinine
450
465
480
495
λ / nm
510
525
0.0 0.2 0.4 0.6 0.8 1.0
Creatinine concentration / mM
TM
Creatinine concentration (Vitros ) / mM
Comparision between human serum creatinine
concentration determined by the VitrosTM
autoanalyzer and by using the IPCCA creatinine
sensor.
0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20
Creatinine concentration (PCCA)/ mM
Conclusions
• New Technology to develop point-of-care
clinical sensors
Blood
ne
i
t
a
cre
ure
a
t ie
coo
s
etc
.
Higher Order
Fantasies !
Sanford A. Asher, Department of Chemistry
PCCA Sensing Array for Glucose, pH,
Recreational Pharmaceuticals and
Alcohol, Stress Hormones, etc.
Subcutaneous Sensors
Visualization of Sensor
Diffraction Through Human Skin
• Fresh Human
Foreskin Harvested
• Fat removed
• Pinned out over
PCCA on dental wax
• Cleared for 5 minutes
with Glycerol
• Imaged with white
light
Subcutaneous Sensors
Subcutaneous Sensors
Macroimaging
Microscope
PCCA
Tissue
PCCA
under
Tissue
Diffraction Measurements Through Tissue
1.8
1.6
PCCA
1.4
Tissue
PCCA Under Tissue
Intensity / au
1.2
1
0.8
0.6
0.4
0.2
0
250
350
450
550
Wavelength / nm
650
750
850
Sanford A. Asher, Department of Chemistry
Outline
• CCA and PCCA Photonic Crystal
Fabrication
• Hydrogel Volume Phase Transitions
• Photonic Crystal Chemical Sensors
-Donnan Potential pH, Glucose and Pb2+
Sensor
-Metal Cation Sensors
-Glucose Sensors
-Creatinine Sensors
-Antibody sensors
BLCA-4
¾ Nuclear matrix protein uniquely expressed
in bladder cancer
¾ BLCA-4 appears to be a very early marker
of bladder cancer
¾ Shown to differentiate patients with
bladder cancer from normal patients with a
high sensitivity (97.7%) and specificity
(100%) in preliminary studies
¾ BLCA-4 Antibody Association Constant:
Ka for the BLCA-4 monoclonal antibody
is 0.4 nmol
¾ BLCA-4 Sequence:
PRFNWLISHTPEGKKKEEREKEKKGENQDLVTRATDRLQTP
VSMESRGLSPGSSKFPPKKTPPHLGMESAITLWQFLLQLLLD
QKHEHLICWTSNDGEFKLLKAEEVAKLWGLRKNKTNMNY
DKLSRALRYYYDKNIIKKVIGQKFVYKFVSFQEILKMDPHA
VEISQLNA
Ionic Residues
Hydrophilic Protein
R: Arginine
D: Aspartic Acid
E: Glutamic Acid
H: Histidine
K: Lysine
Antibody
Cross-linking Sensing Motif
Monoclonal Antibody
Polyclonal Antibody
Antigen
+
+
O
NaOH
C NH2
TEMED
amide-containing
PCCA
O
NHS
C OH
hydrolyzed PCCA
EDC
O
O
C O O N
+
O
amine-reactive PCCA
O
C OH
O
hydrolyzed PCCA
O
C O O N
O
amine-reactive PCCA
O
C N
H
H2N
recombinant Protein G
Protein G-activated PCCA
+
Protein G-activated PCCA
IgG
IgG-containing PCCA
+ BS3
IgG - containing PCCA
Immobilized IgG – containing PCCA
BS3 : Bis(sulfosuccinimidyl) suberate
¾ A water-soluble, homobifunctional N-hydroxysuccinimide
ester (NHS-ester) commonly used to covalently cross-link
antibody to Protein G in preparation of affinity columns
NaO3S
O
O
SO3Na
O
O
SO Na
NaO S
O
O
O
N O C CH
C O NO
2 6 cross-linked to Protein G
IgG
N O C CH C O N
3
3
2 6
O
BS3
O
O
O
O
OO
O
NH
HN
N
C O NH
N 2
NHC
H
C2 CH
CH
2 6 C
2 6
O
Protein G
SO
O 3Na
UV-Vis spectra of CCA-free hydrogel with IgG
incorporated via Protein G
0.3
prior to treatment
after protein G
after IgG
0.25
absorbance (au)
0.2
0.15
0.1
0.05
0
200
250
300
350
400
450
w ave le ngth (nm )
500
550
600
Use of Human Serum Albumin
(HSA) as a Model System for
Antibody Cross-linking
Protein Sensing
Why HSA?
¾Cost
¾Availability
Sensing Response of IPCCA to HSA Protein
¾ Fabricated CCA-free hydrogel and PCCA
•
Used CCA-free hydrogel to spectroscopically monitor the
incorporation of proteins into hydrogels
¾ Functionalized hydrogels
•
Attached monoclonal IgG and polyclonal IgG to PCCA at
a concentration of 0.02 mM
¾ Performed sensing run on functionalized IPCCA
and control PCCA
•
Exposed each gel to concentration of HSA that would be
optimal for 2:1 (Ab:Ag) binding
Sensing Response of IPCCA to HSA Protein
¾ Addition of HSA to an unfunctionalized PCCA gives a
very modest red-shift in diffraction
¾ Addition of HSA to IPCCA gives a modest, yet stable
blue-shift in diffraction
4500
4000
prior to treatment with Ag
3500
after treatment with Ag
4500
intensity (au)
1500
1000
2500
2000
1500
1000
500
500
0
0
-500
-500
400
500
difference spectrum
3000
2000
300
after treatment with Ab
3500
2500
200
prior to treatment with Ag
4000
difference spectrum
3000
intensity (au)
IPCCA
Control PCCA
600
700
w avelength (nm)
Overall red-shift: <1 nm
800
200
300
400
500
600
700
w avelength (nm)
Overall blue-shift: ~5 nm
800
this photonic crystal
technology platform
appears expandable
and useful for
numerous analytes
Acknowledgements
Asher Research Group Members
$: NIH, NCI, NASA and NSF