Probing Protein Adsorption: Total Internal Reflection Intrinsic

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23 Probing Protein Adsorption: Total Internal Reflection Intrinsic Fluorescence R. A. VAN W A G E N E N , S. R O C K H O L D , and J. D. ANDRADE University of Utah, Department of Bioengineering, College of Engineering, Salt Lake City, UT 84112

Interfacial intrinsic fluorescence induced by evanescent wave total internal reflection was developed to study protein ad sorption at solid-aqueous buffer solution interfaces. The tech nique has a number of advantages over conventional meth odologies for the study of adsorption including (1) continuous, real-time sampling with 0.1-s resolution; (2) in situ sensing; (3) application to biomedically

relevant, flat, low surface area

samples; (4) quantitation of the amount adsorbed calculated on the basis of an internal standard; and (5) ability to obtain fluorescence emission spectra of intrinsic tryptophan moieties that are sensitive to local microenvironmental

changes pro

duced during the protein adsorption process. These advan tages are illustrated for bovine serum albumin and γ-globulins adsorbed on hydrophilic

A

quartz.

n understanding of protein adsorption behavior is applicable in numer ous fields including blood-synthetic materials interfaces, macromolec-

ular-membrane interactions, receptor interactions, enzyme engineering, ad

hesion, and protein separation on chromatographic supports. Many methods have evolved to study interfacial adsorption, but no single independent method seems adequate. The ideal technique should produce quantitative, real-time, in situ data concerning the amount, activity, and conformation of proteins adsorbed on well-characterized surfaces. All adsorption techniques are approximations to this optimum. Protein solution depletion via adsorption on finely divided substrates is quantitative, but applicability to low surface area materials of biomedical relevance is often minimal. Adsorption of radiolabeled macromolecules is 0065-2393/82/0199-0351$06.00/0 ©1982 American Chemical Society In Biomaterials: Interfacial Phenomena and Applications; Cooper, S., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

352

BIOMATERIALS: INTERFACIAL PHENOMENA AND APPLICATIONS

quantitative on l o w surface area substrates; however, the presence of an extrinsic label may alter p r o t e i n p h y s i c a l properties and subsequent adsorp tion behavior. A u t o m a t e d e l l i p s o m e t r y can, i n p r i n c i p l e , p r o v i d e i n situ, real-time i n f o r m a t i o n on f i l m thickness and refractive index, but the m i n u t e differences i n substrate, f i l m , a n d buffer refractive indices often p r e c l u d e this approach. M u l t i p l e i n t e r n a l reflection infrared spectroscopy is c o m p l i c a t e d Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on October 11, 2015 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch023

by strong water signals that obscure p r o t e i n a m i d e bands and, w h i l e F o u r i e r transform analysis seems p r o m i s i n g , the interpretation remains difficult and the e q u i p m e n t is expensive. Interfacial p r o t e i n

fluorescence

i n d u c e d by i n t e r n a l reflection evanes

cent wave excitation offers a n u m b e r of advantages over conventional adsorp tion techniques. T h e total i n t e r n a l reflection

fluorescence

( T I R F ) concept

was o r i g i n a l l y p a t e n t e d b y H i r s c h f e l d (1 ) and a p p l i e d to p r o t e i n adsorption by H a r r i c k a n d L o e b (2). Since t h e n T I R F has b e e n u t i l i z e d i n a l i m i t e d n u m b e r of investigations to study the adsorption of extrinsic plasma proteins o n q u a r t z (3, 4),

fluor-labeled

h a p t e n - p r o t e i n conjugates (5), and p o l y -

d i m e t h y l s i l o x a n e films (6). O u r p r e l i m i n a r y d e v e l o p m e n t of T I R F also e m ployed covalently b o u n d

fluorescein

isothiocyanate ( F I T C ) as an extrinsic

fluor (7, 8); however, research has i n d i c a t e d that F I T C l a b e l i n g of a l b u m i n is labile a n d also alters the proteins chromatographic and electrophoretic properties (9). E x t r i n s i c labels p e r se are not objectionable as l o n g as the presence of the l a b e l can be s h o w n not to alter the b i o c h e m i c a l and p h y s i c o c h e m i c a l p r o p e r t i e s of the m o l e c u l e b e i n g s t u d i e d . H o w e v e r , the attrac tiveness of i n t r i n s i c T I R F is that the difficulties i n l a b e l i n g and c o n f i r m i n g the inertness of the l a b e l are c o m p l e t e l y o b v i a t e d . W e report here the successful d e v e l o p m e n t of i n t r i n s i c , interfacial p r o t e i n

fluorescence

based on t r y p

tophan excitation. T h e advantages offered b y i n t r i n s i c T I R F are illustrated w i t h data for a l b u m i n a n d 7-globulin adsorption on quartz.

Principles of Internal Reflection Fluorescence W h e n light of w a v e l e n g t h λ , t r a v e l i n g i n a m e d i u m of refractive index n encounters a second m e d i u m of refractive index n (n < n{), it undergoes total i n t e r n a l reflection i f the angle of i n c i d e n c e , Θ, exceeds the critical angle 6 , w h e r e 0 =sin~ (n /n ). T h e rectangular coordinate system of F i g u r e 1 illustrates this p h e n o m e n o n . T h e electric f i e l d vectors may be resolved into components p a r a l l e l , Ey, a n d p e r p e n d i c u l a r , £ , to an optical plane d e l i n eated by the i n c i d e n t a n d reflected beams. T h e superposition of i n c i d e n t and reflected radiation establishes a standing wave n o r m a l to the reflecting inter face as i l l u s t r a t e d i n F i g u r e 1. T h e electric f i e l d a m p l i t u d e has a nonzero value E ° at the surface, w h i c h t h e n decays exponentially into the less dense m e d i u m . T h e p e r p e n d i c u l a r p o l a r i z a t i o n - m o d e electric f i e l d a m p l i t u d e at b

C

2

c

1

2

2

1

±

In Biomaterials: Interfacial Phenomena and Applications; Cooper, S., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.


23.

VAN W A G E N E N E T A L .

353

Probing Protein Adsorption

Ζ

η, > n Θ >e

2

c

Figure 1. Left: Schematic of the coordinate system at a totally reflecting interface separating two media of refractive index η and n . Right: Standing wave pattern and exponential decay of the electric field vector into the less dense medium, 2. γ

the interface (10),

E °, ±

2

can be r e p r e s e n t e d as 0

2 cos θ

=

1

m

[1 " (n ) P/2

y

21

2

{

)

w h e r e n \ = (n /rii). A n e l e c t r o m a g n e t i c d i s t u r b a n c e t e r m e d the evanescent wave penetrates the rarer m e d i u m to a finite d e p t h . It has a wavelength λ and is continuous w i t h the sinusoidal f i e l d of the standing wave, but the electric f i e l d a m p l i t u d e Ε decreases e x p o n e n t i a l l y w i t h distance f r o m the surface ζ as 2

2

Ε = £ ° e x p [~z/d ]

(2)

p

T h e effective wave p e n e t r a t i o n d e p t h d is the distance z, w h e r e the electric field a m p l i t u d e Ε has decayed to e~ of its surface value E°, as given b y E q u a t i o n 3. p

l

2ττη

άρ=

ι

[sin 6 - (n f] A 2

2l

l

( 3 )

T h e value of d decreases at shorter wavelengths, greater index m i s m a t c h i n g p

(ni »

n ), a n d i n c i d e n t angles a p p r o a c h i n g the critical angle (Θ—»0 ). 2

Interfacial

C

fluorescence

p r o v i d e s an excellent means of s t u d y i n g p r o t e i n

adsorption. T h e m a x i m u m e n e r g y available for excitation is l o c a l i z e d w i t h i n a few h u n d r e d angstroms of the surface w h e r e most of the p r o t e i n is concen-


354


trated. C o n s e q u e n t l y , the vast majority of the fluorescence signal arises f r o m adsorbed p r o t e i n m o l e c u l e s . A n adsorbed interfacial f i l m of refractive index n complicates the analysis of interfacial electric f i e l d d i s t r i b u t i o n . G e n e r a l l y , 3

the f i l m is assumed to b e m u c h t h i n n e r than d a n d the f i l m electric f i e l d p

d i s t r i b u t i o n is assumed to b e u n i f o r m . T h e f i e l d d i s t r i b u t i o n for the parallel c o m p o n e n t d e p e n d s strongly o n n , b u t the p e r p e n d i c u l a r f i e l d c o m p o n e n t 3

E, Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on October 11, 2015 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch023

±

is not affected b y t h e t h i n f i l m a n d remains d e f i n e d b y E q u a t i o n 1.

M a c r o m o l e c u l a r properties may alter the f i l m refractive index n , w h i c h i n 3

t u r n c o u l d affect t h e available excitation energy d i s t r i b u t i o n a n d r e s u l t i n g flourescence signal levels. C o n s e q u e n t l y , the p e r p e n d i c u l a r c o m p o n e n t E

±

was used i n this research. (See Refs. 8, 10, 11, a n d 12 for m o r e details o n T I R F principles.) T h e study o f m a c r o m o l e c u l a r adsorption o n t h i n p o l y m e r films s h o u l d also be feasible. A d s o r p t i o n isotherms can be obtained o n any nonfluorescing p o l y m e r that can b e d e p o s i t e d i n t h i n - f i l m f o r m o n quartz. T h e o n l y l i m i t a tion is that the f i l m b e n o n a b s o r b i n g at the excitation wavelength a n d exhibit minimal

fluorescence.

T h i n films of d e p t h d can be s t u d i e d i f [2πά/λ]

< 0.1

and i f the f i l m attenuation i n d e x κ is less than 0.1 (JO). T h i n films o n quartz substrates can t h e n b e c h a r a c t e r i z e d b y other surface analytical techniques. M o l e c u l a r interaction w i t h radiation is p r o p o r t i o n a l to the radiation intensity, a n d thus to the square of the electric f i e l d vector, Ε . 2

λ

Equations

1-3 were used to generate F i g u r e 2 w h i c h illustrates the variation of E

2

±

ζ for both i n t r i n s i c a n d extrinsic T I R F at t h e q u a r t z - a q u e o u s interface. I n t r i n s i c

fluorescence

(d = 1040 A ) than is extrinsic p

with

electrolyte

is m o r e localized i n the interfacial region fluorescence

u s i n g F I T C (d = 2235 A ) . p

Experimental Figure 3 illustrates the experimental configuration. The light source (A) was a 200-W mercury-xenon high-pressure lamp, and the monochromator (B) selected the fluorescence excitation wavelength. A 10-cm focal length quartz lens (L) reduced the beam diameter and, in conjunction with a front-surface, silvered mirror (M), redi rected the light through a UV-polarizing filter (Oriel 2732) (0) oriented to pass the perpendicular component of radiation. Light entered one face of the quartz dove tail prism (Q), and illuminated approximately 1/cm of the central prism face contacting the aqueous buffer in the flow cell (F). The prism was UV-grade quartz (Markson Science Inc.)—3-cm wide, 9-cm long, and 2.9-cm thick with face angles of 70° to the base. The flow cell base was a 3-cm wide, 9-cm long, and 1.3-cm thick block of marine-grade aluminum alloy (5086-Hll) anodized flat black after machining rec tangular slit-flow ports at the surface of each end. Aluminum was chosen because of its ease of machining, passivity to cleaning solvents, and good thermal conductivity. The anodized film endowed the cell base with excellent inertness to aqueous saline solutions. The flow cell base and prism were separated by a Silastic rubber medicalgrade polydimethylsiloxane gasket 0.05 cm thick. The effective cell flow field was 7.9 x 2.1 X 0.05 cm. Flow rates during sample injection andflushranged from 1.5 to 2.0 mL/s. Flow at the sampling area was laminar (Reynolds numbers 140-190) and well established (flow development length 0.12 cm). Flow cell surface area and 2





2.8

I

I

0

1000

I

I

I

3000

I

5000

I

ι

I

7000

PENETRATION DEPTH , Ζ (A) Figure 2. Exponential decay in (E ) with distance ζ from the reflecting interface. ±

2



356


Figure 3. Schematic of experimental system. Light source (A), excitation and emission monochromators (B and C., respectively), PMT (D), preamplifier (Ε), flow cell(F), constant temperature recirculator(G), syringe pump (H), photon counter (I), light tight enclosure (J), digital disc storage (K), quartz lens (L), mirrors (M), beam stop (N), excitation and emission polarizing filters (O and P, re spectively), trapezoidal quartz prism (Q), chart recorder (R), ana copper heat exchanger base plate (S).

volume were 34.1 cm and 0.83 cm , respectively. The amount of bulk solution depletion due to protein adsorption on the cell walls was not measured, but order of magnitude calculations suggest that it could be significant for C less than 0.1 mg/mL. However, this result depends on how much protein is assumed to adsorb to the flow cell walls. Flow cell base temperature and all solutions entering the cell were main tained at 37° ± 1°C by an attached copper base plate (S) thermally linked to a constanttemperature bath (G). A fraction of the interfacial fluorescence penetrated the prism and entered an emission monochromator (C). Both monochromators (B) and (C) (Jobin Yvon, H-10) were used with 16-nm bandpass resolution, the only exception being for spectra scans where the emission monochromator bandpass was 4 nm for optimal resolution. Fluor escence quantitation was accomplished by using an RCA 8850 photomultiplier tube (D) linked to an Ortec photon counting system with the following components: preamplifier 9301 (E), amplifier-discriminator 9302, photon counter 9315 (I), sam pling control module 9320, and digital data storage using a 5V4-in. floppy disk on an Apple II Plus Computer (K). Digital signals were converted to analog (Ortec 9325) and displayed on a Pharmacia 481 strip chart recorder (P). Well-definedflowparam eters were maintained by a Sage Instruments 341 syringe pump (H). An acrylic, light tight housing (J) and beam stop (N) helped keep extraneous light signals at low levels. All solutions were made with analytical-grade reagents and low-conductivity (6 megohm) water purified by a combination of reverse osmosis, ion exchange, activated-carbon adsorption, and microfiltration. Phosphate-buffered saline (PBS) (0.145M NaCl, 2 x 10" M K H P 0 , 8 x 1 0 M Na HP0 ) had a pH of 7.3 and 2

3

B

4

2

4

_4

2

4



23.

VAN WAGENEN ET AL.


357

osmolarity of 310 mosmol. All solutions were deaerated initially, but because this procedure had no effect on fluorescence intensity, deaerating was not a routine procedure. Proteins were obtained from Miles Laboratories as bovine serum albumin (BSA) monomer standard Fraction V (81-028-1-P338) and bovine 7-globulins Fraction II (82-041-2-1086). L-Tryptophan (Matheson Coleman & Bell), 0.3 mg/mL in PBS, was used as an intrinsic fluorescence experimental reproducibility standard. The quartz prism surface was cleaned prior to each experiment in the following sequence: (1) 5 min soak in 2% (v/v) Microclean, (2) 15-min soak in dichromatesulfuric acid (25/mL Manostat Chromerge chromic acid in 4.1 kg of concentrated sulfuric acid), (3) thorough rinse in low-conductivity, filtered water, (4) rinse in filtered, absolute ethanol, (5) 5-min vapor degreasing in Freon TES-ethanol azeotrope vapor, (6) 2-min radiofrequency (RF) glow discharge at 30 W tuned RF power (Tegal, Plasmod), 200 μπι mercury pressure oxygen plasma, and (7) 10-min purge in ultrapure (99.999%) oxygen. All surface cleaning and assembly were carried out under Class 10,000 clean room conditions to minimize particulates and enhance experi mental reproducibility. Figure 4 illustrates schematically the time course of a TIRF experiment. The PBS background is due to scatter in the primedflowcell and stray light passed by the wide monochromator slits. The fluorescence signal intensity is expressed as counts per unit time above the PBS background. Injection of a tryptophan standard was used as an internal reference point for comparing the reproducibility of each experiment. With high recording speeds (10 cm/s), short count times (0.1 s), and rapid solution injection (1.5-2.0 mL/s), the speed with which the tryptophan bulk signal reached its equilibrium count level was determined. This procedure typically required 1-2 s after the first hint of signal increase. Similarly, the time required to remove all of the tryptophan from the cell at 2.0 mL/s was about 2 s. The cell priming volume, includ ingflowports, was 1.5 cm . No indication that tryptophan adsorbed irreversibly was observed, since count rates before and after the standard were identical. Protein was introduced under the same sampling and flow conditions. At bulk concentrations greater than 0.5 mg/mL, a small signal step of 1-2 s, N was immedi ately evident on the recorder at high speed and short sampling times (see Figure 5). The adsorption signal then rapidly developed on top of N . With the exception of injecting and flushing out protein solutions, all adsorption and desorption occurred under nonflow conditions. After reaching equilibrium signal level for any particular bulk protein concentration, the bulk protein was removed via a 50-mL flush of PBS at 2 mL/s. A rapid incremental signal drop N occurred due to removal of the bulk solution contribution followed by a slower decrease in signal as the protein molecules desorbed from the surface (see Figure 4). Protein adsorption dynamics could then be monitored. Alternatively, additional protein at a greater bulk concentration could be added in a stepwise manner to determine the adsorption isotherm. Solutions of higher bulk protein concentration were added when the equilibrium plateau signal had remained stable for 10-15 min. Since the time required to reach the plateau was generally about 20 min, the total elapsed time between different concentrations was typically 40 min. This entire step isotherm was determined on a single surface rather than the more classical and lengthy approach of obtaining one datum point at a particular C on a single fresh surface. Emission spectra of adsorbed protein were taken following the PBS flush. In this way only the spectra of adsorbed protein were determined. These spectra were then compared to bulk solution spectra of nonadsorbed protein obtained with the same equipment, but with the TIRF prism and cell replaced by a conventional transmission fluorescence bulk cell. Tryptophan amino acid emission spectra were recorded both in a conventional spectrofluorometer bulk cell and with the standard in the TIRF flow cell. 3

Bi

B

B

B



CO

LU

CO

>-

ο ο

LU CO \ CO

ο

PBS BASELINE

PROTEIN

t B

U

L

K

TIME (MIN)

C

BUFFER FLUSH

A

PROTEIN

t

BULK=Ν Β

BUFFER

Figure 4. Schematic of TIRF data during a typical experiment. A tryptophan standard initiates each experiment. The stippled area represents adsorbed protein producing N counts per unit time.

BACKGROUND SIGNAL

mg/ml

L-TRYPTOPHAN STANDARD

0.3



23.


w

3000

Ο \ CO

2000

Z> Ο

1000

359


ο

0

2

4

6

TIME ( S E C ) Figure 5. Fluorescence data for y-globulin on quartz. The bulk signal N arises during the first 1-2 s of flow.

B

Results and Discussion Interfacial

fluorescence

signals d i d not y i e l d direct information on the

amount of a d s o r b e d p r o t e i n . T h i s lack of quantitation appears to be the major weakness of the m e t h o d . C a l i b r a t i o n studies have b e e n attempted (6), but the ideal solution w o u l d be a second, i n d e p e n d e n t quantitative technique such as F T I R or use of u n a l t e r e d r a d i o l a b e l e d p r o t e i n . A n i n d e p e n d e n t calibration m e t h o d is n o w b e i n g d e v e l o p e d . O u r approach was to use the

fluorescence

b a c k g r o u n d signal 2V ema B

nating f r o m evanescent w a v e - e x c i t e d p r o t e i n i n the b u l k solution as an internal calibration signal. C o n s i d e r the 3000-A decay d e p t h of F intrinsic

fluorescence

2

for the

case of F i g u r e 2. T h e evanescent wave penetration

d e p t h was d i v i d e d into 30 lamellae, each 100 A thick and 1/cm . A d s o r p t i o n 2

was d e f i n e d as those m o l e c u l e s o c c u p y i n g the first l a m e l l a adjacent to the surface. M o l e c u l e s i n L a m e l l a e 2 - 3 0 w e r e c o n s i d e r e d to have b u l k properties and to constitute the b u l k signal. T h e total

fluorescence

signal was p r o p o r

tional to the area u n d e r C u r v e 2 of F i g u r e 2, but it was u n e q u a l l y w e i g h t e d to the first f e w l a m e l l a e , w h e r e most o f the e n e r g y was available to excite the majority of the m o l e c u l e s . T h e area of each l a m e l l a was integrated and expressed as f i e l d i n t e n s i t y units squared ( F I U ) . T h e first l a m e l l a had 2.11 2

FIU

2

w h i l e the s u m of L a m e l l a e 2 - 3 0 was 9.9 F I U . A t a b u l k p r o t e i n 2

concentration C

B

of 1.0 m g / m L , each b u l k l a m e l l a contained 1 X 10~ g of 9

p r o t e i n . P r o t e i n i n the b u l k lamellae (29 X 0.001 μg/cm ) was excited b y 9.90 2

F I U and p r o d u c e d N counts, w h i l e adsorbed p r o t e i n Γ ^ g / c m ) was excited 2

2

B

by 2.11 F I U

2

and produced N

A

counts. B u l k signal N

B

was resolved f r o m

adsorbed p r o t e i n signal N d u r i n g the first 1-2 s of p r o t e i n injection and flush A

(See F i g u r e s 4 a n d 5). N

B

was accurately d e t e r m i n e d for C

C o n s e q u e n t l y , for the quantitation of N

B

B

^ 0.5 mg/mL.

at lower b u l k concentrations,


a

360


linear plot of N

B

a C

B

as a f u n c t i o n of (0.5 < C ^ 4.0) m g / m L was extrapolated to B

of 0 m g / m L b e t w e e n (0.0 < C < 0.5) mg/mL. T h e equality of E q u a t i o n B

4 results a s s u m i n g that i n b o t h b u l k a n d adsorbed states, a certain quantity of p r o t e i n excited b y a g i v e n a m o u n t of energy ( F I U ) p r o d u c e d an equal 2

amount of

fluorescence

signal p e r u n i t t i m e , that is, the q u a n t u m yields of


b u l k and a d s o r b e d p r o t e i n w e r e e q u a l . (C ) B

(0.001 μg/cm lamella) (29 lamellae) (9.90 F I U ) _ N counts/unit t i m e 2

2

B

(Γ) (2.11 F I U ) 2

N

A

( 4 )

counts/unit t i m e

T h e correction for b u l k p r o t e i n concentrations represented b y C . Surface concentrations B

other than 1.0 m g / m L is

Γ ^ g / c m ) were calculated on 2

the basis of E q u a t i o n 4 a n d p r e s e n t e d as a function of t i m e or b u l k concen tration C

B

for a d s o r p t i o n - d e s o r p t i o n dynamics or adsorption

isotherms,

respectively. T h i s research a n d its associated quantitation rests on several assump tions. F i r s t , l a m p p o w e r o u t p u t was assumed to be invariant w i t h times comparable to the course of an e x p e r i m e n t . T h i s assumption was reasonable since source intensity m o n i t o r i n g v i a b a c k g r o u n d counts Ν for 6-8 h showed no significant drift i f the l a m p a n d p h o t o n c o u n t i n g system were e q u i l i b r a t e d for 1 h p r i o r to an e x p e r i m e n t . C o u n t rates Ν were t y p i c a l l y 500 counts and the c o u n t i n g statistics e r r o r rarely e x c e e d e d V N by m o r e than several (0.1/s) percent. S e c o n d , the excited b u l k concentration and thus N are t i m e invar iant. T h i s assumption is p r o b a b l y correct i f C is not r e d u c e d by adsorption w i t h i n the flow c e l l and i f no p h o t o b l e a c h i n g occurs. A l s o , our fast s a m p l i n g data i n d i c a t e d that for b o t h t r y p t o p h a n standard and p r o t e i n solution, a b u l k signal c o m p o n e n t c o u l d be resolved d u r i n g the first 1-2 s f o l l o w i n g sample injection. T h e p r o t e i n adsorption signal intensity t h e n b u i l d s on top of this. A s s u m i n g that C is established m o r e slowly by diffusional processes alone, some interfacial time-variant d i s t r i b u t i o n function w o u l d have to be i n c o r p o rated into E q u a t i o n 4. S u c h a function w o u l d initially lower the interfacial concentration a n d reduce Γ. T h i r d , q u a n t u m yields of adsorbed- a n d b u l k fluorescing species are i d e n t i c a l . T h i s is p r o b a b l y the most questionable assumption, p a r t i c u l a r l y i f conformational changes occur f o l l o w i n g ad sorption. Q u a n t u m y i e l d determinations of adsorbed species are p l a n n e d i n the future. F o u r t h , light scattered f r o m b o t h adsorbed molecules a n d b u l k B

B

B

molecules w i t h i n the evanescent zone does not generate significant b u l k p r o t e i n solution fluorescence. T h i s assumption w o u l d have the effect of re d u c i n g N , i n c r e a s i n g i V , a n d c o n s e q u e n t l y l o w e r i n g Γ . F i f t h , the adsorbed f i l m is 100 A thick, that is, it is the first l a m e l l a . A s s u m i n g that the adsorbed f i l m and thus the first l a m e l l a was 50 or 150 A thick, the calculated value of Γ w o u l d be a l t e r e d b y less than ± 5 % . Sixth, N c o u l d not be d e t e r m i n e d A

B

B


23.

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361


accurately b e l o w a C o f 0.5 m g / m L . C o n s e q u e n t l y , a plot of N as a function B

B

of ( 0 . 5 < C < 4 . 0 ) m g / m L was l i n e a r l y extrapolated to zero i n the range B

(0 < C < 0.5) m g / m L to y i e l d values of N . B

B

F i n a l l y , w e assume no occurence of interfacial p h o t o c h e m i s t r y . This was generally true for the l i g h t intensities a n d times u t i l i z e d i n o u r research as illustrated i n C a s e 1 o f F i g u r e 6 for adsorbed b o v i n e 7-globulin. F o l l o w i n g Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on October 11, 2015 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch023

p r o t e i n i n t r o d u c t i o n , the surface concentration Γ rose to an e q u i l i b r i u m value, a n d r e m a i n e d t i m e invariant even w h e n additional p r o t e i n of the same b u l k c o n c e n t r a t i o n was injected into the c e l l . H o w e v e r , w h e n the light source i n t e n s i t y was increased b e y o n d a c r i t i c a l level b y either increasing l a m p p o w e r o r r e p l a c e m e n t w i t h a fresh m e r c u r y - x e n o n l a m p b u l b , the equilibrium

fluorescence

signal was not attained. Signal intensity peaked

q u i c k l y a n d t h e n d e c a y e d c o n t i n u o u s l y . T h i s p h e n o m e n o n is referred to as Case II b e h a v i o r a n d is i l l u s t r a t e d i n F i g u r e 6. T h i s b e h a v i o r is not s u r p r i s i n g i n that m a n y investigators have detected photoeffects

u n d e r c o n v e n t i o n a l spectrofluorometric conditions.

Studies

c o n d u c t e d o n the U V - i r r a d i a t i o n of l y s o z y m e (13) a n d L-glutamate d e h y drogenase (14) r e v e a l e d a c o n c o m i t a n t loss i n e n z y m e activity a n d fluores cence e m i s s i o n w i t h i r r a d i a t i o n t i m e . T h e p r i m a r y photoeffects were destruc-

Figure 6. Schematic of typical TIRF data with no photoeffects (Case 1) and with photoeffects causing a time variable signal (Case 2) for bovine y-globulin adsorbed on hydrophilic quartz.


362



tion of t r y p t o p h a n residues a n d the appearance of S H groups (13) as disulfide bonds w e r e d i s r u p t e d . E n z y m a t i c inactivation may have resulted f r o m ter tiary structure loss d u e to scission of the four d i s u l f i d e bonds c r o s s - l i n k i n g the single p o l y p e p t i d e c h a i n of l y s o z y m e . T h e d e s t r u c t i o n of aromatic a n d s u l f u r - c o n t a i n i n g amino acid residues and r e s u l t i n g loss of h e l i c a l structure f o l l o w i n g 68 m W U V - i r r a d i a t i o n also have b e e n r e p o r t e d for a l b u m i n (15). H o w e v e r , i n o u r research, signal i n stabilities postulated to result f r o m p h o t o c h e m i c a l effects w e r e always associ ated w i t h 7-globulin, but not a l b u m i n adsorption. A l s o , o u r calculated max i m u m U V light p o w e r was t y p i c a l l y m u c h less, that is, several megawatts at most. O n the basis of t r y p t o p h a n content alone, 25 residues i n 7-globulin and two i n B S A , the l i k e l i h o o d of p h o t o c h e m i c a l susceptibility i n 7-globulins is great. B o t h proteins have at least 16 d i s u l f i d e bridges that m a i n t a i n structural integrity, but b o t h m o l e c u l e s appear to have substantial chain f l e x i b i l i t y . A t wavelengths o f 2 7 0 - 2 9 0 n m or greater, aromatic residues and d i s u l f i d e bonds of proteins adsorb w e l l , a n d because t r y p t o p h a n has the lowest t r i p l e t state energy of all the a m i n o acids, light adsorption by other residues results i n excited states that may migrate to specific t r y p t o p h a n moieties v i a a n o n radiative transfer m e c h a n i s m . O n e p r i m a r y oxidation reaction of photoexcited t r y p t o p h a n is electron transfer to an acceptor m o l e c u l e a n d C 2 - C 3 b o n d cleavage of the i n d o l e r i n g y i e l d i n g N - f o r m y l k y n u r e n i n e (16). T h e adsorption i s o t h e r m for b o v i n e 7-globulin on h y d r o p h i l i c quartz is illustrated i n F i g u r e 7. T h e e q u i l i b r i u m plateau concentration of 3.60 μg/cm is substantially h i g h e r than most other 7-globulin adsorption data on silica and glass substrates, that is, several tenths of a m i c r o g r a m p e r square c e n t i m e t e r (17, 18). T h i s result p r o b a b l y occurs because T I R F is an i n situ t e c h n i q u e y i e l d i n g values of Γ o b t a i n e d i m m e d i a t e l y after r e m o v a l of the b u l k signal. T h i s is i n contrast to most adsorption methods e m p l o y i n g an extensive p r e q u a n t i t a t i o n buffer rinse that may cause d e s o r p t i o n of a loosely b o u n d , r a p i d l y d e s o r b i n g layer(s) (6), the presence of w h i c h w o u l d not be generally d i s c e r n i b l e . 2

Q u a n t i t a t i o n of Γ was made on the basis of b u l k b a c k g r o u n d counts N d e t e r m i n e d by a discrete step change i n fluorescence intensity o c c u r r i n g either as p r o t e i n was i n i t i a l l y i n t r o d u c e d or d u r i n g the first few seconds of the buffer flush. A l t e r n a t i v e l y , N , and thus Γ, c o u l d be d e t e r m i n e d later d u r i n g the flush sequence, for e x a m p l e , at 50 m L total flush v o l u m e rather than at 4 m L . T h i s w o u l d be a closer a p p r o x i m a t i o n to sample w a s h i n g conditions d e s c r i b e d for the m o r e c o n v e n t i o n a l approaches. T h e result w o u l d be a h i g h e r value of 2V , a lower value of Ν , a n d a value of Γ m o r e than 50 percent lower than o u r data. In situ fluorescence appears to give larger values of Γ because c o n v e n t i o n a l methods r e q u i r e longer rinse times w h i c h i n t u r n remove substantial amounts of loosely adherent m o l e c u l e s . P r e l i m i n a r y data d e t e r m i n e d for r e p r o d u c i b i l i t y studies at a C of 1.0 m g / m L on clean, h y d r o p h i l i c quartz gave an e q u i l i b r i u m plateau adsorbed B

B

B

λ

B


23.


363


4

3 00

Έ 2 ο


en

0

-I

0

1

2

3

4

CB (mg ml") Figure 7. Adsorption isotherm for bovine y-globulin on hydrophilic quartz at 37°C. 1

value of 1.69 ± 0 . 1 6

μ^τη

2

(η = 4). N o t s u r p r i s i n g l y , step i s o t h e r m and

discrete adsorption i s o t h e r m m a x i m a w e r e substantially different. T h e con formational state of an adsorbed p r o t e i n may be a function of surface resi dence t i m e , solution activity, and n u m b e r of adsorbed neighbors. Because adsorption is o n l y p a r t i a l l y reversible, C

B

cannot be increased or decreased

to obtain a p a r t i c u l a r value of Γ, that is, hysteresis effects do occur. S u c h hysteresis b e h a v i o r has b e e n w e l l d o c u m e n t e d i n adsorption experiments on w e l l - d e f i n e d c h r o m a t o g r a p h i c substrates (19).

T h e application of T I R F to

both d e s o r p t i o n a n d adsorption d y n a m i c s as w e l l as b o t h approaches

to

isotherm d e t e r m i n a t i o n s h o u l d make the study of hysteresis effects more p r o d u c t i v e i n the f u t u r e . A significant decrease o c c u r r e d i n adsorbed p r o t e i n signal as reflected i n Γ at b u l k concentrations e x c e e d i n g 2 m g / m L . T h i s decrease was also evident in o u r earlier extrinsic T I R F adsorption studies w i t h 7 - g l o b u l i n - F I T C fibrinogen-FITC

(8),

a n d also has

been

observed

and

b y others s t u d y i n g

7-globulin a d s o r p t i o n (3). T h e actual cause is as yet unresolved. H o w e v e r , this significant d r o p i n fluorescence a n d thus i n apparent Γ may be due to fluorescence

q u e n c h i n g at h i g h surface concentrations

or p r o t e i n

con

formational changes a n d s u b s e q u e n t l o w e r i n g of q u a n t u m y i e l d that may occur w i t h increased surface p a c k i n g density. M o r r i s s e y et al. (18, 20) shown

that 7-globulin

does

undergo

substrate-induced

have

conformational

changes d u r i n g a d s o r p t i o n o n silica, a n d the degree of conformational altera tion appeared to d e p e n d on surface concentration. Since our isotherms are d e r i v e d i n a b u l k concentration step-increase fashion, h i g h e r b u l k concentrations

(>2

mg/mL) were not evaluated for

several hours after p r o t e i n first contacted the quartz surface. T h i s delay may


364



p r o v i d e sufficient t i m e for 7-globulin molecules to change conformationally i n such a m a n n e r tha q u a n t u m y i e l d s fall a n d the c o r r e s p o n d i n g fluorescence signal drops or d e s o r p t i o n of a l t e r e d p r o t e i n occurs. Transmission circular d i c h r o i s m ( C D ) studies have shown major conformational changes d u r i n g the adsorption a n d subsequent activation of H a g e m a n factor on quartz (21). Recently, c o n f o r m a t i o n a l changes associated w i t h the adsorption of a l b u m i n , 7-globulin, a n d f i b r i n o g e n o n to c o p o l y p e p t i d e a n d silicone substrates were r e p o r t e d (22). T h r e e stages c h a r a c t e r i z e d b y reversible adsorption, i r r e v e r s i ble a d s o r p t i o n , a n d slow structural alteration and d e s o r p t i o n of d e n a t u r e d p r o t e i n m o l e c u l e s w e r e r e p o r t e d . H o w e v e r , the t i m e course of these events was many hours to several days. M o r r i s s e y et al. saw no significant effect of t i m e on the c o n f o r m a t i o n of a d s o r b e d 7-globulin (21 ). A l t e r n a t i v e l y , an o r d e r e d , stable array of macromolecules may evolve at the surface w i t h i n c r e a s i n g t i m e and/or b u l k concentration. Since the ex citation light is p l a n e - p o l a r i z e d , the decay i n e m i s s i o n signal may be a reflec tion of p r e f e r e n t i a l o r i e n t a t i o n of 7-globulin molecules w i t h t i m e or enhanced surface p a c k i n g densities. F l u o r e s c e n c e e m i s s i o n polarization studies s h o u l d tell us m o r e about this i n the future. F i g u r e 8 illustrates the adsorption i s o t h e r m for B S A on h y d r o p h i l i c quartz. T h e value of Γ is n o w on the o r d e r of 0.1 μg/cm , and the amount adsorbed is d i r e c t l y p r o p o r t i o n a l to b u l k concentration C w i t h no h i n t of plateau saturation b e l o w 5.0 m g / m L . T h e s e surface concentrations are c o m parable to data r e p o r t e d b y others (23-27) for B S A adsorption o n silica and glasses. A t a C of 1 m g / m L , r e p o r t e d values range f r o m 0 . 0 2 - 0 . 2 μg/cm , d e p e n d i n g o n p H , t e m p e r a t u r e , i o n i c strength, a n d substrate. O u r value of 0.08 μg/cm at the same C is i n t e r m e d i a t e i n this range. T h e s e results parallel o u r qualitative findings for B S A - F I T C adsorption o n quartz (8). A l b u m i n a d s o r p t i o n continues to o c c u r at concentrations exceeding those r e q u i r e d for m o n o l a y e r coverage. T h i s result is at odds w i t h most research, w h i c h indicates an e q u i l i b r i u m coverage of several tenths of a m i c r o g r a m p e r square c e n t i m e t e r at a C greater than several m i l l i g r a m s p e r m i l l i l i t e r . H o w e v e r , the means of o b t a i n i n g these isotherms are not d i r e c t l y c o m p a r able w i t h o u r T I R F data, w h e r e reversible d e s o r p t i o n is m o r e accurately based on r a p i d l y r e m o v i n g o n l y the b u l k signal p r i o r to reversible d e s o r p t i o n of " p e r i p h e r a l p r o t e i n . " T h e c o n t i n u a l b u i l d u p of B S A w i t h increasing C may result f r o m h y d r o p h o b i c b o n d i n g b e t w e e n molecules c o m p r i s i n g differ ent layers. T h e surface of the B S A m o l e c u l e may have h y d r o p h o b i c patches (28), w h i c h may i n t u r n facilitate m u l t i l a y e r b i n d i n g at increasing c o n c e n trations v i a h y d r o p h o b i c b o n d i n g . 2

B

2

B

2

B

B

B

Interfacial p r o t e i n fluorescence is an i n situ m e t h o d that can p r o v i d e real t i m e data w i t h a r e s o l u t i o n of 0.1 s. T h i s t e c h n i q u e is a major advantage i n that the p r o t e i n a d s o r p t i o n - d e s o r p t i o n d y n a m i c s may be d e t e r m i n e d w i t h out r e s o r t i n g to sample m a n i p u l a t i o n p r i o r to analysis. F i g u r e 9 illustrates a d s o r p t i o n - d e s o r p t i o n d y n a m i c s for b o t h B S A a n d 7-globulin at b u l k e q u i molar concentrations of 6.67 μΜ/L. T h e 7-globulin r e q u i r e d 40 m i n to reach In Biomaterials: Interfacial Phenomena and Applications; Cooper, S., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.



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an e q u i l i b r i u m o f 1.80 μg/cm , w h i l e the B S A adsorbed to an e q u i l i b r i u m 2

value o f 0.06 μg/cm i n a p p r o x i m a t e l y 8 s, as illustrated i n F i g u r e 10. T h e 2

initial d e s o r p t i o n d y n a m i c s w e r e e x t r e m e l y r a p i d for both proteins (see F i g ure 11). D u r i n g the first 3 s , B S A d e s o r b e d at a rate of 24 ng/cm /s, a n d w i t h i n 2

6 s, most o f the p r o t e i n h a d d e s o r b e d . H o w e v e r , a small amount o f B S A always s e e m e d

to r e m a i n i r r e v e r s i b l y adsorbed (7), that is, about 0.01

μg/cm . S u c h r a p i d rates m i g h t s i m p l y have b e e n the result o f b u l k solution Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on October 11, 2015 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch023

2

fluorescence.

H o w e v e r , i f this w e r e true, the same T I R F behavior w o u l d

occur on all surfaces, w h i c h clearly was not the case, as illustrated already for 7-globulin on h y d r o p h i l i c quartz. V i r t u a l l y no data exist i n the literature w i t h w h i c h w e m a y c o m p a r e this adsorptive behavior o c c u r r i n g i n the first few seconds of surface contact. D y n a m i c e l l i p s o m e t r y is too slow, and r a d i o l a b l e d p r o t e i n e x p e r i m e n t s are i n a p p r o p r i a t e d u e to r e q u i r e d washing steps. T h e i n i t i a l d e s o r p t i o n rate for 7-globulin was linear for the first 30 s at 6.7 ng/cm /s. T h i s r a p i d d e s o r p t i o n m a y be d u e to the presence o f a r e 2

versibly a d s o r b e d p e r i p h e r a l layer o f 7-globulin. T h e existence o f this a d sorbed 7-globulin layer has b e e n r e p o r t e d i n other T I R F research as w e l l (3, 6). A transition t i m e o f several m i n u t e s separates two essentially linear regions of 7-globulin d e s o r p t i o n . T h e second linear desorption phase has a rate o f 0 . 0 6 7 ng/cm/s over a 6 0 - m i n p e r i o d , that is, a factor one h u n d r e d times slower than the i n i t i a l d e s o r p t i o n rate. A f t e r m o r e than 1 h of d e s o r p t i o n , a substantial a m o u n t o f 7-globulin remains " i r r e v e r s i b l y " adsorbed o n the quartz surface. B a s e d o n the second phase rate, at least 13 days w o u l d be r e q u i r e d for all of the 7 - g l o b u l i n to desorb u n d e r these conditions. H o w e v e r , over periods e x c e e d i n g 2 h , the d e s o r p t i o n rate was not c o m p l e t e l y linear, but was w e a k l y e x p o n e n t i a l . C o n s e q u e n t l y , i r r e v e r s i b l y adsorbed layers of 0.80

0.60

Έ

0.40

U

0.20

υ

0.00

BSA

-0.40 -2

8

18

28

38

48

TIME (SEC)

Figure 10. Adsorption dynamics for 6.67 μΜ/L BSA (0) and y-globulin (·) on hydrophilic quartz at 37°C.



23.

VAN WAGENEN ET AL.

367


Figure 11. Desorption dynamics for 6.67 μΜ/L BSA (o) and y-globulin (·) on hydrophilic quartz at 37°C. B S A a n d 7-globulin appear to exist o n h y d r o p h i l i c quartz, a n d the surface molar ratio o f B S A : 7-globulin is 0.02 p i c o m o l / c m : 8.0 picomol/cm after 1 2

2

h of d e s o r p t i o n . Since d e s o r p t i o n was p e r f o r m e d statically, such proteins might c o n t r i b u t e to an excess i n b u l k solution, a n d " i r r e v e r s i b l e " p r o t e i n then w o u l d not b e i n e q u i l i b r i u m w i t h a true zero b u l k concentration, b u t this does not seem to b e a significant effect. P r e l i m i n a r y experiments indicate that i f a d d i t i o n a l r i n s i n g occurs d u r i n g the desorption phase, that is, re establishing zero for C , the d e s o r p t i o n rate does not change appreciably. B

Perhaps the single greatest advantage of T I R F is its ability to d e t e r m i n e an adsorbed p r o t e i n e m i s s i o n s p e c t r u m , w h i c h is illustrated i n F i g u r e 12 for bulk t r y p t o p h a n a n d a d s o r b e d 7-globulin a n d B S A . I n a l l three cases, the emission spectra are b r o a d (300-460 n m ) , a n d several have shoulders at wavelengths e x c e e d i n g 350 n m , w h i c h is understandable for b o t h proteins, particularly 7-globulin. T h e heterogeneity of the proteins as w e l l as the local m i c r o e n v i r o n m e n t of particular t r y p t o p h a n moieties c o n t r i b u t e to a w i d e range of fluorescence emissions. C e l l u l o s e acetate electrophoresis i n d i c a t e d that B S A a n d b o v i n e 7-globulin w e r e 9 9 % p u r e . H o w e v e r , b o v i n e s e r u m F r a c t i o n II 7-globulins c o m p r i s e a variety of different i m m u n o g l o b u l i n types, and each of these may b e f u r t h e r s u b d i v i d e d into variations i n the F

a b

section.

T h e local t r y p t o p h a n m i c r o e n v i r o n m e n t p r o b a b l y contributes the most d i versity to the e m i s s i o n spectra. A t least three distinct spectral classes of t r y p t o p h a n exist, one b u r i e d d e e p i n nonpolar regions of the p r o t e i n and two at the surface, one c o m p l e t e l y and one o n l y partially exposed to the aqueous e n v i r o n m e n t (29). S u c h spectral classification for the 26 tryptophans i n 7-globulin and the two tryptophans i n B S A can b e e m p l o y e d to elucidate the



368


295

335

375

415

455

495

λ ( nm)

Figure 12. Fluroescence emission spectra for evanescent wave excited "interfacial" L-tryptophan (A), adsorbed bovine y-globulin (B), and adsorbed BSA (C). e m i s s i o n spectra of b o t h proteins b e t w e e n 3 0 0 - 3 6 0 n m . T h e a m o u n t and k i n d of fatty acid c o m p l e x e d to B S A molecules affect the λ a n d intensity of t h e i r e m i s s i o n s p e c t r u m (30). N o t e the tyrosine e m i s s i o n peak λ at 305 n m for B S A . A l b u m i n is one of the few proteins that e x h i b i t tyrosine fluo rescence p r i m a r i l y because of its l o w q u a n t u m y i e l d and the existence of several q u e n c h i n g m e c h a n i s m s (31 ). Μ Α Χ

Μ Α Χ

T h e shoulders at wavelengths e x c e e d i n g 360 n m are difficult to e x p l a i n , particularly for the t r y p t o p h a n case. T r y p t o p h a n molecules w e r e possibly concentrated at the q u a r t z - b u f f e r interface i n some k i n d of weak, but suf ficiently close association to create resonance fluorescence p h e n o m e n a ex h i b i t i n g several peaks b e t w e e n 3 6 0 - 4 6 0 n m . S i m i l a r peaks exist for b o t h B S A and 7-globulin, a n d since the b a c k g r o u n d spectra w e r e subtracted to leave the c o r r e c t e d spectra of F i g u r e 12, e n v i s i o n i n g h o w an i n s t r u m e n t a l artifact c o u l d cause the p h e n o m e n a is difficult. B u l k fluorescence spectra taken i n a conventional A m i n c o B o w m a n s p e c t r o f l u o r o m e t e r showed no such s h o u l d e r behavior for L - t r y p t o p h a n , B S A , or 7-globulin; however, the spectral resolu tion was not as g o o d as that i n the T I R F system. B u l k fluorescence emission spectra o b t a i n e d w i t h a c o n v e n t i o n a l 1-cm quartz c e l l and the same T I R F optical and e l e c t r i c a l e q u i p m e n t p r o d u c e d data comparable to those obtained i n the s p e c t r o f l u o r o m e t e r . C o n s e q u e n t l y , the shoulders appear to be i n d e p e n d e n t of the i n s t r u m e n t a t i o n , a n d the strong p o s s i b i l i t y exists that c o n formational changes are r e f l e c t e d i n a l t e r e d fluorescence e m i s s i o n spectra that accompany the a d s o r p t i o n of B S A a n d 7-globulin on quartz.


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In summary, T I R F offers a number of advantages over conventional techniques for studying macromolecular adsorption. First, it is an in situ method yielding real time data with resolutions of 0.1 s. Second, the geometry of the prism flow cell system makes possible the study of factors affecting protein adsorption, that is, interfacial shear stress, temperature, buffer properties, etc. Third, fluorescence emission spectra of adsorbed macromolecules Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on October 11, 2015 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch023

are, in principle, capable of providing information on macromolecular conformational changes accompanying adsorption. This fact is particularly true for the intrinsic fluorescence emission of tryptophan residues that are extremely sensitive to local microenvironments within and upon the protein surface. Finally, by selectively labeling one type of protein, studying competitive protein adsorption with all of the previously mentioned advantages should be possible. This theory is, of course, predicated upon the use of a fluor that does not alter the physicochemical and biochemical properties of the macromolecule to which it is attached.

Acknowledgments We thank Joel M . Harris for constructive advice, and acknowledge N I H Grant HL-18Î519-05 for financial support of this research.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

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R E C E I V E D for review January 16, 1 9 8 1 . A C C E P T E D June 3 , 1 9 8 1 .


Probing Protein Adsorption: Total Internal Reflection Intrinsic

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