Surface plasmon immunoassay - OSA Publishing

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of dinitrophenyl (DNP) and keyhole limpet hemocyanin (KLH) antibodies in blood serum samples, indicate that the SPI, in addition to providing a simple and fast ...
Surface plasmon immunoassay Eduardo Fontana, R. H. Pantell, and Samuel Strober

The most sensitive existing assays used to determine antibody levels in blood serum samples require a tracer material, e.g., radioisotope, fluorofore, or enzyme, to identify the specific analyte. Surface plasmon spectroscopy has been applied recently as a no-label technique for the assay of specific antibody solutions with the antigen proteins immobilized on a metal surface. It is found that the metal surface configuration originally proposed for the surface plasmon immunoassay (SPI) is unstable and unsuitable for the assay of specific antibodies in a large mixture of proteins such as in a blood serum. Nevertheless, by properly designing the metal surface structure, the SPI can be made an extremely practical device. Preliminary results for the assay of dinitrophenyl (DNP) and keyhole limpet hemocyanin (KLH) antibodies in blood serum samples, indicate that the SPI, in addition to providing a simple and fast measurement, is comparable with existing approaches, such as radioimmunoassy or enzyme-linked immunosorbent assay both in sensitivity and specificity.

1. Introduction

The most sensitive procedures used to detect or quantify the amount of antibody or antigen molecules in biological fluids are generally based on labeling of the protein molecule under study.' The labeled molecule is bound to the protein of interest so that the basic properties of both molecules are retained. For the detection of antibodies, the corresponding specific antigen is usually immobilized in a solid phase, e.g., a plastic test tube, which after a sequence of washing and drying steps is exposed to the antibody solution. After the immunological reaction reaches an equilibrium, another sequence of washes is used to remove nonreactants, and the amount of labeled material remaining is measured. In solid phase radioimmunoassay (RIA),1 the protein to be measured is tagged using radioactive isotopes as labels, so the radioactivity left in the test tube after the final stage gives a measure of the antibody bound to the solid phase. In the enzyme-linkedimmunosorbent assay (ELISA), 2 ,3 the antigen coated test tube after reaction with the solution containing

When this work was done all authors were with Stanford University, Stanford, California 94305; S. Strober was in the Department of Medicine, Division of Immunology &Rheumatology, and the other authors were in the Electrical Engineering Department. Eduardo Fontana has now returned to Federal University of Pernambuco, Department of Electronics & Systems C. P. 7.800, Recife, Pernambuco 50.741, Brazil. Received 27 February 1989. 0003-6935/90/314694-11$02.00/0.

©1990 Optical Society of America. 4694

APPLIED OPTICS / Vol. 29, No. 31 / 1 November 1990

antibodies, is rinsed and a second antibody-enzyme conjugate solution is added to the test tube. The second antibody reacts only with the antibodies of interest. The final step is to add a solution to the test tube that is converted to a colored substance in presence of the enzyme left in the test tube. The degree of conversion is proportional to the amount of antibodyenzyme conjugates bound to the serum antibodies in the solid phase. Thus, one can visually observe a color reaction or measure the solution optical absorptance in the ultraviolet so that a quantitative result is obtained. The above procedures are specific and sensitive, with both having comparable limits of detectability of approximately 10-9 grams/ml.1-3 Radioactive substances, required for RIA, can be hazardous, and so present interest has focused on alternative nonradioactive techniques, such as ELISA. Common to the above procedures is the necessary labeling step, the use of somewhat sophisticated detection systems for quantitative measurements and, in the case of ELISA, the use of light sources operating in the ultraviolet wavelength region. In addition, because measurements are taken after completion of the immunological reaction, these methods require several hours to obtain quantitative results. In this work, we describe a procedure that can be applied to quantitative determination of specific proteins in blood serum. This method is based on an optical technique called surface plasmon spectroscopy (SPS),4 which has been proposed in the literature as a means for monitoring the dynamics of the antigenantibody immunological reaction. 5 6 As will be seen, the surface plasmon immunoassay (SPI) does not re-

quire labeling of the proteins under study, it can be

IL LJ

C-) z

-J

x

wL Iu

I £ >0

0s

DIELECTRIC

8S

MEDIUM I E-field amplitude Fig.1. Surface plasmon at an interface separating two semi-infinite regions, one of which is a metal. The maximum amplitude occurs at the interface and falls off exponentially within each region. The wave has an electric field normal to the boundary, and propagates along the x-direction with a phase velocity smaller than the velocity of light in the dielectric medium. At optical wavelengths the metal has a permittivity Ethat is negative; whereas, the of the dielectric is positive. This condition is necessary to establish the surface plasma wave.

LASER BEAM

> n2

PRISM

2

-

x METAL

DIELECTRIC MEDIUM

implemented using a simple light source such as a HeNe laser or even a light emitting diode (LED), and it can produce fast quantification of specific proteins in a mixture of different proteins. In addition, SPI provides a sensitivity comparable to the procedures previously described. II. Surface Plasmon Spectroscopy The excitation of a surface plasmon (SP) can take place in the interface between a metal and a dielectric medium. The electric field of the wave is normal to the boundary and has its maximum at the interface, decaying exponentially within each region as illustrated in Fig. 1. Because of its confinement to the interface, the SP parameters, such as amplitude, velocity and damping, are sensitive functions of the properties of the boundary between the two media. The object of SPS is the excitation and detection of these waves and the measurement of their parameters so that information can be obtained about the properties of the two regions having the common interface indicated in Fig. 1. A simple prism coupling scheme, illustrated in Fig. 2, has been used to couple a laser beam to a SP.7 The purpose of the prism is to provide the conditions for the existence of an evanescent wave within the dielectric medium adjacent to the metal film, thus creating a slow wave on the metal-to-transparent medium interface. The evanescent wave will exist as long as the incidence angle of the probing beam is kept above the critical value O,, where

o =sin-'Qh),

(1)

where n1 and n 2 are the prism and transparent dielectric medium refractive indices, respectively, and n2 > n 1. The critical angle given by Eq. (1) corresponds to a value above which total internal reflection would occur

RMUF=

7rZ

I

///0

Z F/

,

-

-

d

500A

-

I

Fig.2. Prism method to couple a laser beam to a SP. The probing laser beam polarized in the x-z plane strikes the prism base coated with a metal film a few hundred angstroms thick. The prism provides the conditions for the existence of an evanescent wave within the dielectric medium if the inequality n, > n2 is satisfied. Surface plasmon excitation is observed as a dip in the reflected intensity, and the minimum occurs when the phase velocity of the incident wave in the plane of the interface equals the SP phase velocity.

in the absence of the thin metal film. The incoming wave must have a field component in the plane of incidence (defined as the plane formed by the wavevector and the surface normal) so that an electric field component normal to the prism base plane is obtained. The angular dependence of the reflectance, defined as the ratio between intensities exiting and entering the prism shown in Fig. 1, respectively, has the resonant behavior illustrated in Fig. 2. The minimum in the resonance curve occurs for an incidence angle 0, such that the velocity matching condition, V = VP,

(2)

is satisfied, where Vx=

-

nl

sinG,

(3)

is the phase velocity of the incoming beam parallel to the prism-to-metal interface, c is the velocity of light in vacuum and vsp is the SP phase velocity at the wavelength of the incident beam. The resonant angle is a sensitive function of the optical constants of the two contacting media, and on resonance all or part of the incident beam energy is transferred to the SP. Metal film thickness determines the fraction of incident light energy converted to the SP, and for silver, the opti1 November 1990 / Vol. 29, No. 31 / APPLIED OPTICS

4695

O 0

X

LASER BEAM

-

0

a

9--

, §s4-PRISM MEDIUM l

lAg

LASER BEAM 4-PRISM MEDIUM

4

500 A THICK

(a)

Ag

500 A THICK

(C)

ANTIGEN PROTEIN FILM ANTIBODIES IN SOLUTION

0

,.-11

ANTIGENS IN SOLUTION

y

-=< I, REFLECTED INTENSITY UPON EXPOSURE TO ANTIBODY SOLUTION

R 2

I

R --- -- - - -- - --

2

0

9SP z

z LU 0

(d)

R 2 R

(e)

aL

w 0c UJ

0SP

0

TIME

Fig.3. Surface plasmon immunoassay (SPI) principle. (a) The silver film is exposed to an aqueous solution containing antigen proteins. The resonance curve for a metal-solution configuration will be shifted to larger angles with respect to the one obtained in air because of a larger value for the solution refractive index, as illustrated in (b). Next, the metal surface with the antigen coating is placed in contact with a solution containng antibodies, which upon binding will further shift the resonance curve as indicated in (d). Measuring the reflected intensity vs time at the angle 0= Osp where Osp is the resonance angle before antibody attachment, yields the plot depicted in (e). The initial slope of the reflectance vs time plot is proportional to the antibody concentration in solution.

mum energy transfer occurs for a thickness of 500 A at = 5461 A.8 The width of the resonance curve is related to the damping of the SP due to optical absorption within the metal and to radiation losses back into the prism. At X = 5461 A, a typical width of 0.50 is measured for a silver film 500 A thick in contact with air.8 Due to the sharp nature of the SP resonance curve, it is possible to use this effect to measure the thickness of very thin films. A dielectric coating on the metal layer shown in Fig. 2 alters the position of the resonance minimum and this effect has been used by Pockrand 9 to determine the thickness of Langmuir-Blodget organic monolayer assemblies 26 A thick on silver films. The sensitivity of his apparatus could resolve thicknesses down to angstrom dimensions. Roughness on the order of tens of angstroms present on the metal surface can also modify the location, width and minimum reflectance of the resonance curve, as compared to the measured curve in a smooth metal sur-

face.' 0 The information obtained from these mea4696

APPLIED OPTICS / Vol. 29, No. 31 / 1 November 1990

sured differences is useful in determining the metal surface roughness parameters."' The SPS approach is also applicable for monitoring immunological reactions. The usefulness of the technique has been demonstrated in the literature for the measurement of pure antibody solutions. 5' 6 The basic principle involved in applying the SPS technique to measure antibody concentration is illustrated in Fig. 3. A glass substrate having the same refractive index as the prism shown in Fig. 2 is used for the metal film deposition and is brought into optical contact with the prism base with the use of an index matching fluid having the same refractive index of both prism and substrate. The metal film is then immersed in an aqueous medium containing antigens [Fig. 3(a)]. Because the solution refractive index is larger than the refractive index of air, the total internal reflection condition will occur at an angle larger than the corresponding value for a prism-air interface, shifting the resonance curve as illustrated in Fig. (3b). Antigen

adsorption on the metal forms a monolayer, causing a

further shift in the resonance curve to larger angles. The incidence angle is tuned close to the new resonance value and the solution containing antigens is replaced by a solution of antibodies specific to the antigen molecules [Fig. 3(c)]. Binding of the antibodies to the antigen molecules increases the deposited optical thickness on the metal surface producing an additional shift in the resonance angle. In Fig. 3(d), the effect of antibody binding on the resonance curve is shown for three different times T = 0, T = T1, and T = T2 , with T 2 > T1 > 0. Measuring the initial rate of change of reflectance provides information on the antibody concentration in solution [Fig. 3(e)]. This is the procedure that has been considered in the literature. 5 6 However, in most practical situations the solution not only contains specific antibodies but also a much larger number of nonspecific proteins. Thus, -it is important to demonstrate that this technique is able to differentiate antibodies of interest in a mixture of proteins, and with a sensitivity comparable to existing assays. In addition, it has been observed in the literature that the resonance curve becomes significantly broader than predicted when a silver surface is used in contact with solution,6 thereby significantly decreasing the sensitivity of the SPS approach. As will be shown in the next section, the broadening is caused by instabilities of the metal surface upon exposure to an aqueous medium. In this paper we extend the technique as follows: Specificity is demonstrated, i.e., that a given antibody can be identified in a serum containing a mixture of proteins. A stable silver layer is obtained, thereby maintaining maximum sensitivity. The sensitivity of the procedure is measured. The thickness of a protein monolayer is measured, and this measurement is related to the protein molecular dimensions. Ill.

Experiment One of the objectives of this work is to demonstrate that the SPI can be applied to measure specific antibodies directly in blood serum. The latter consists of a variety of different types of proteins amounting to a total concentration of approximately 80 mg/ml, where antibodies of a given specificity are usually a small fraction of the total protein content in the serum sample. The antiserum was obtained from mouse blood immunized using dinitrophenyl conjugated to keyhole limpet hemocyanin (DNP-KLH) as immunogen. The antiserum containing DNP and KLH specific antibodies was characterized for the anti-DNP content using the ELISA method with a minimum detectable concentration in the dilution range 1:10,000-1:100,000. The antigen proteins used to coat the silver surface were KLH, DNP conjugated to bovine serum albumin (DNP-BSA) for the antibody measurements, and bovine gamma globulin (BGG) to test the specificity of the method. Rabbit anti-BGG was also used in the experiments for additional specificity checks. The

He-Ne laser * 4 -JI

*POLARIZER ROTATION STAGE (I MIN RESOLUTION) METAL FILM ER

ME

Pi IO1TODIODE B I

fl)

PHOTODIODE A STRIP CI-iART RECORDER

B* Ratio

A A/B M 0_0e er

Fig.4. Experimental apparatus for measuring antibody concentration. A rotation stage provides the relative movements of the prism with respect to the incoming laser beam, around an axis normal to the incidence plane. The light source is a He-Ne laser tube operating at X = 6328 A and the glass substrate is coated with a Ag film 500 A thick. The laser beams entering and exiting the prism are collected by a pair of photodetectors, and the ratio between the photocurrents is obtained by the ratiometer, thus eliminating fluctuations from the laser source. The ratio signal is read out and/or sent to the strip chart recorder where the reflectance vs time curves can be plotted.

purified BGG and rabbit anti-BGG proteins were purchased from Sigma Chemical Company, St. Louis, MO. The experimental apparatus for the adsorption measurements is shown in Fig. 4. The laser beam from a He-Ne laser source operating at a wavelength X = 6328 A, passes through a polarizer before striking the prism. A beam splitter sends approximately 50% of the incident beam to photodetector B and the main beam passes through a halfwave plate before striking the prism-substrate-metal film. The halfwave plate is used to place the beam polarization parallel to the incidence plane. The reflected beam is collected by photodetector A and the ratio between the signals from A and B is either read directly or measured by a strip chart recorder. A rotation stage having minimum resolutions of 3 min of arc and 2 s of arc for gross and fine adjustments, respectively, is used to perform the angular movements around an axis perpendicular to the incidence plane. Also illustrated in Fig. 4, is the cavity where the solutions containing different proteins are placed during the experiment. The cavity is made of plexiglas and has a volume of 400 Al. Figure 5 1 November 1990 / Vol. 29, No. 31 / APPLIED OPTICS

4697

is a detailed drawing of the arrangement for bringing the cavity in contact with the film. The cavity is pressed against the metal surface and an 0-ring is used in between for sealing.

METAL FILM

GLASS SUBSTRATE PRISM Fig.5. Details of the contact between metal surface and cavity. The 0-ring is sandwiched between metal surface and cavity. The cavity volume is 400 ,ul.

A. Metal Surface Stability In the early experiments of this work, the metal films were made of silver 530 A thick directly deposited on glass substrates as illustrated in Fig. 6(a). We chose to use silver because it has the smallest loss tangent (ratio between the imaginary and real part of E)in the visible range relative to any other metal which, in turn, means that the reflectance curve measured in thin films of this material has a very sharp resonance. However, when these films were exposed to saline solution prior to any of the protein deposition, significant surface modifications, attributable to poor bonding of the silver to glass,' 2 were observed as changes in reflectance of the laser beam. In addition, for the particular configuration of our experiment, the direct contact between the 0-ring and metal surface, as illustrated in Fig. 5, causes a damage ring on the metal surface. Water molecules penetrate through the damage ring

CIRCULAR DEPOSIT OF THIN METAL FILM

O-RG

>

_____CAVITY DAMAGE RING Ag

530 A THICK

4-

-GLASS

GLASS SUBSTRATE

(a)

SUBSTRATE

.I

I

_v''I

CAVITY

Ag

500

= 30

SUBSTRATE

A THICK

A THICK

(b)

Cr

30

A THICK

(C)

Fig.6. Thin metal film stability design. (a) Silver film 500 A thick on top of glass substrate, with cavity 0-ring pressed against the surface. The damage ring is caused by removal of part of the Ag surface which adheres to the rubber 0-ring; (b) same as in (a) with a 30 A Cr layer under the Ag film; (c) surface damage is eliminated by depositing circular metal spots on the glass substrate having diameter smaller than the 0-ring diameter, thus avoiding contact between metal surface and 0-ring. 4698

APPLIED OPTICS / Vol. 29, No. 31 / 1 November 1990

diffusing along paths of weak bonding under the Ag film. As a result, regions on the Ag surface can either detach or become loose from the glass substrate. In either case, a rough metal surface will be produced whose effect is to shift and broaden the resonance curve,6 10 thus causing instabilities. Consequently, a silver film deposited directly on the glass substrate is unsuitable for the immunoassay design. The stability of the metal surface was improved by predepositing a thin chromium layer between the glass substrate and silver film as illustrated in Fig. 6(b). The choice of chromium was based on its well-known good adherence to glass and most of metals, the degree of adherence increasing in proportion to the chromium thickness.13 Since chromium has a strong optical absorption in the visible range,1 4 we were limited to use of a maximum thickness of 30 A of this material, so as to avoid a significant broadening of the resonance with a corresponding loss in sensitivity. Using this procedure, metal surfaces stable for 1 h upon exposure to a saline solution could be obtained, but the stabiity was still not adequate to measure low antibody concentrations. Surface stability was greatly improved by avoiding direct contact between the 0-ring and the metal surface, as illustrated in Fig. 6(c). Instead of covering the whole glass substrate with the metal films, circular spots of these films, each having a diameter smaller than the corresponding 0-ring diameter, were deposited on the substrate, thus eliminating any damage to the metal surface. Under these conditions, the metal films thus produced were stable for a minimum of several days in contact with solution and were suitable for measuring the lower range of concentrations. We also verified that the protein films left on these multilayer surfaces after an adsorption experiment could be removed by means of a Q-tip with the surface immersed in trichloroethane followed by acetone and methanol rinsing. Thus, the surface could be restored to its original state and reused in additional experiments. B. Antigen Coating of the Metal Surface In the experiments performed thus far, three types of antigen proteins were used to coat the Ag surface: keyhole limpet hemocyanin (KLH), bovine gamma globulin (BGG), and dinitrophenyl-bovine serum albumin conjugate (DNP-BSA). Because the SPS method can detect very thin coatings deposited on a metal surface, dynamic processes related to adsorption or desorption of even very small amounts of proteins can be studied. Antigen coating consisted of exposing the clean Ag surface to a solution containing 1.0 mg/ml of the antigen protein diluted in a 0.154-M NaCl solution. The laser beam intensity reflected from the prism base was monitored as a function of time during protein adsorption. After a certain time, a steady state regime for adsorption is reached for which no further change in reflectance is observed and -4 ml of saline solution is applied to the cavity for rinsing, with the coated Ag

KLH

o.o 10 F

,

7

°

RINSING

/BGG

, 0.6 0

.5

RINSINGI

DNP-BSA

0.3

d

2

. 2

0 .1

0

10

20

30

40

50

60

70

Time (minutes) Fig.7. Differential reflectance, obtained by subtracting the measured value at time t from the corresponding value at t = 0, vs time for a Cr-Ag bilayer exposed to KLH, BGG and DNP-BSA protein solutions all at 1 mg/ml. The laser beam incidence angle was fixed close to the resonance value for the metal in contact with a clean saline solution. The rinsing induced desorption is indicated as a drop in reflectance for the KLH and DNP-BSA cases.

surface remaining wet during rinsing. The time dependence of the reflectance, for the three classes of proteins studied in this work is shown in Fig. 7. These curves were obtained by fixing the laser beam incidence angle close to the resonance value for the metal surface in contact with saline solution. The stock protein solutions were diluted upon insertion into the cavity yielding a final protein concentration of 1 mg/ml. From Fig. 7, one can notice that the KLH and DNP-BSA adsorptions yield the largest and smallest changes in reflectance, respectively, with the BGG adsorption curve being intermediate with respect to the other two. When the KLH and DNP-BSA surfaces were rinsed by flowing the saline solution through the cavity, a slight decrease in reflectance was observed, as shown in Fig. 7. The magnitude of the shift indicates a maximum 10% desorption of proteins from the surface with rinsing. This desorption can cause some nonspecific binding in the measurement of specific antibodies from serum samples, and this will be discussed in the next section. A typical plot of the angular dependence of the resonance curve prior and after adsorption is shown in Fig. 8(a), for the case of a KLH coating. Both curves have the same shape and the one coated with the KLH protein is up-shifted -I' with respect to the one measured prior to deposition. The fact that the resonance curve retains the same shape after coating, indicates a good uniformity of the KLH film. Also shown in Fig. 8(a) are the theoretical predictions for the data with both data and theoretical curves plotted as the ratio between intensities within the prism medium. The calculated ratio between intensities was obtained using the procedure outlined in Refs. 4 and 7, assuming a 1 November 1990 / Vol. 29, No. 31 / APPLIED OPTICS

4699

Table 1. Experimental Parameters

i.0

X = 6328 A

Wavelength 0.6

a)

0.6

U

Silver complex permittivity' Chromium complex permittivitya

EAg = -16.84 - iO.628 ECr = -6.219 - i31.21

Prism refractive index Saline solution refractive index Protein film refractive index Cr film thickness

n = 1.643 n = 1.332 n = 1.5 dCr = 30 A dAg = 500 A

Ag film thickness

0

0.4

a

cc

Ref. 14.

0.2 Table II. 0.0 54

62

60

58

56

Angle

Average Thickness and Standard Deviation for Different Proteins Adsorbed on Ag Surface

64

(deg)

Average thickness (A) Standard deviation (A) Number of measurements

(a)

KLH

BGG

DNP-BSA

86 12 8

57 1.2 3

20 2.5 3

1.0 0.9 0.8 0.7 0.6

0.5 rc

0.4 0.3

E

_

0. 2_

0.0

_

55

56

L.

57

_

......

58

59

...

60

Angle

61

_

62

.

63

64

65

(deg)

(b) Fig.8. (a) Resonance curves before and after KLH deposition. The dots and continuous curves are the data and theoretical predictions, respectively. Calculated curves were obtained using the parameters given in Table I. The measurements were performed on glass slides that received more than one metal deposition, with the films chemically removed between depositions with an NH 3 0H solution. (b) Data and theoretical prediction for the metal-saline solution resonance curves, for a glass substrate that received a single metalization.

prism-Cr-Ag-saline solution multilayer configuration with the values of optical constants and thicknesses listed in Table I. The prism and saline solution refractive indices were obtained from the measured total internal reflection angle for the prism base without and with saline solution sitting in the cavity, respectively. One can notice from Fig. 8(a) that the data curves are broader than predicted and this effect varied between different slides. Broader data curves were obtained for glass slides that received more than one metal deposition, with the metal films being chemically removed with a NH 3 0H solution between depositions. On the other hand, reflectance curves mea4700

APPLIED OPTICS / Vol. 29, No. 31

1 November 1990

sured on glass slides that received a single metal deposition produced better agreement with theory, as illustrated in Fig. 8(b). We concluded that an alloy formed between the glass surface and the thin Cr layer, which could not be removed using the chemical treatment, resulting in broader curves, due to roughness on slides receiving multiple metalizations. Other factors, such as a Ag film thickness smaller than the assumed value of 500 A, can also contribute for the broadening of the data curves shown in Fig. 8(a). We calculated the coating thickness for each of the antigens investigated in this work from the total shift in resonance angle after protein adsorption, as an average of values obtained from different experiments and the results are shown in Table II. The calculations were performed using the first-order expression ob-, tained by Pockrand 9 for the change in the SP wavevector due to the deposition of a thin dielectric layer on the metal surface. The formulation of Pockrand is valid for a metal film coated with a thin dielectric layer with the former in contact with the prism base. Because the 30-A thick Cr film used in our bilayer structures does not significantly change the Ag resonance position, the Cr underlayer has a negligible effect in the results. This justifies neglecting the presence of Cr, and using the formulation of Pockrand in the present estimates, Pockrand's result can be written as:

Akp

=

(,)

12 (1

(n)2)

_n2 n2

/2.II\/A2\

+

+

2 __l

E'l

_

_in_2

2lrd

-el n2

where Aksp is the shift in the SP wavevector, d is the thickness of adsorbed protein having refractive index n,, n, is the solution refractive index, and e' is the real part of the Ag complex permittivity. The above expression can be rewritten as a function of the angular shift of the resonance minimum referred to the prism

HEMOCYANIN MOLECULAR STATES

35A

1/10

1/1

SIDE VIEW

TOP VIEW

-------------------------------

190 A 190A 190A

I

I ---------------A

300A

I'~~~~~~~~~~

A ---

1/2 1/1 1 1/2 Fig.9. Molecular states of gastropod hemocyanin, which include Keyhole limpets, found under neutral pH conditions. The basic structure, labeled as 1/1 is a hollow cylinder having a pair of collar substructures. The 1/10 molecule is a well-defined fraction of the 1/1 molecule, which retains one molecular residue of the collar substructure. The 1/2 and 1 1/2 molecular structures are similar to the 1/1 molecule, having heights of 1/2 and 11/2 of the correspondingheight of the 1/1 molecule, respectively. Themoleculardimensions were extracted from the electron micrographs shown in Figs. 9 and 11 of Ref. 17.

medium AO by noticing that ksp 27r/Xnl sin~sp, where Osp is the SP resonance angle prior to adsorption, and thus Aksp (2nr/X)nl cos~spAO. This gives:

O=

c

1/2 1 -

nj coSspnjl'l/

:1)

(n)2)(n2

ncJ Jn + I (lT s2)

X

(4)

Thus, the angular shift can be calculated from the known optical constants of the prism, organic layer, solution and metal film for a given wavelength which are given in Table I for X= 6328 A, and the observed value of the resonance angle in solution prior to protein adsorption. The protein molecule refractive index was estimated from the known difference of 0.0002 in refractive indices between a 1 mg/ml protein solution and a pure aqueous solution.' 5 Assuming that the protein solution refractive index equals the sum of 1 November 1990 / Vol. 29, No. 31 / APPLIED OPTICS

4701

0.12

Table Ill. Heights for Each of the Molecular Structures Depicted In Fig. 9, and for the Case of a Cylindrical (A) or Flat Surface (B) of the Molecule Parallel to the Metal Surface

Molecular state

1 1/2 1/1 1/2 1/10

Proportion in Solution (%)

Case A

(A)

Case B

(A)

5

97

184

55 20 20

97 97 35

123 61.5 35

Average = 84.6

96.2

a) ca a)

cc 0 'a

'E

relative masses of solution and protein weighted by the respective refractive indices, a value of 1.5 is obtained for the refractive index of a protein molecule. The average value of 86 A for the KLH coating thickness is indicative of the size of this protein molecule. Hemocyanin from keyhole limpets (Megathura crenulata) exists in four aggregation states under neutral pH conditions.' 6 The basic structure, labeled as 1/1 in Fig. 9, is a hollow cylinder having two collar substructures, as observed from electron micrographs, with the dimensions indicated in Fig. 9.17 It has also been shown that the states 1/10, 1/2, 1/1 and 1 1/2, represented in Fig. 9, occur in proportions 20%, 20%, 55% and 5% under neutral pH conditions, respectively, with the basic structure 1/1 having a molecular weight of 9 X 106.17

To calculate the protein coating thickness, we first calculate the equivalent height for each of the molecular structures shown in Fig. 9 and then we average these heights in proportion to the molecular states adsorbed to the surface. In a first approximation, it is assumed that the molecular states in solution remain in the same proportion upon adsorption to the surface. The average height, h can be written as h = V/A, where V is the molecular volume and A is the area of a rectangular section parallel to the silver surface encompassing the projection area of the protein molecule. The average KLH thickness was calculated for two protein configurations on the metal surface, namely, with either the cylindrical (Case A) or flat surface (Case B) parallel to the metal surface. It was also assumed that the 1/10 molecule binds to the surface with the corresponding flat plane parallel to the silver surface, which is reasonable since more binding sites are available for adsorption in this configuration. The average height for each of the molecular states is given in Table III, and the average thickness values calculated for cases A and B are 84.6 and 95 A, respectively. These two figures encompass and agree rather well with the measured value of 86 A, so that the above model yields an excellent prediction for the SPS data. An ellipsoid of rotation with major and minor axes of 140 A and 40 A, respectively, has been proposed to represent the molecular structure of a BSA molecule. 18 Since addition of a small molecule such as DNP is not expected to cause any significant structural modification, we assume the above structure to represent the

DNP-BSA molecule. Assuming the DNP-BSA mole4702

APPLIED OPTICS / Vol. 29, No. 31 / 1 November 1990

40

60

100

Time (min)

Fig.10. Differential reflectance vs time for KLH coated Cr-Ag surface exposed to antiserum solution containing KLH and DNP antibodies, for varying ten-fold dilutions between 1:10-1:10,000. For this set of measurements the incidence angle was tuned close to the metal-KLH-saline solution resonance condition.

cules adsorbing with a large number of binding sites, i.e., with the major ellipsoid axis parallel to the silver surface, an average thickness value of 21 A is obtained which is in close agreement with the measured value of 20 A. For the BGG molecule, because of its three fragment structure, it is somewhat more difficult to define an equivalent geometry to apply our model. Nevertheless, the measured value given in Table II is in good agreement with reported values in the literature.5 C. Antibody Measurements Antiserum measurements were obtained using serum samples of mice immunized with the DNP conjugated to KLH. For the evaluation of KLH antibodies, the incidence angle was fixed close to the resonance value for the prism-Cr-Ag-saline solution configuration and the antiserum was introduced in the cavity. Figure 10 illustrates the time dependence of the differential reflectance, obtained by subtracting from the measured reflectance the corresponding value at t = 0, for varying ten-fold dilutions of the antiserum in the range 1:10-1:10,000. Even though an apparently small change is observed in the scale of Fig. 10 for a dilution of 1:10,000, this change is clearly measured on the chart recorder. The detectability of the SPI at 1:10,000 is comparable to the one obtained with existing assays. To ensure that the curves illustrated in Fig. 10 did represent the adsorption of KLH antibodies, we have made a number of specificity tests. As mentioned in Sec. II.B, the desorption of antigen proteins from the metal surface upon rinsing can give rise to some non-

specific adsorption of proteins from an antiserum. It

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(b) Fig.11. (a) Differential reflectance as a function of time for three different coatings exposed to a 140-fold diluted antiserum solution containing the KLH and DNP antibodies. (b) Differential reflectance vs time for a BGG coating exposed to two different antibody solutions.

may happen that the antibody content is much smaller than the amount of nonspecific serum proteins, and this would make it difficult to differentiate the specific event. Nevertheless, this nonspecific effect can be minimized by using. a 1-mg/ml BSA solution as dilutent and as the rinsing agent. The BSA solution should remain a few minutes in the cavity prior to antiserum insertion. Using this procedure, open sites left in the metal surface will be occupied by the BSA molecules, greatly reducing nonspecific adsorption. An alternative to this procedure would be the use of nonimmunized serum in all antiserum dilutions to cover the possible open sites on the metal surface. Another possibility would be to perform the measurements under dry conditions, wherein the antigen coated metal surface is exposed to the antiserum for a standard time interval of a few minutes and then the surface is

rinsed and dried. Reflectance readings before and after reaction would be compared, with the rinsing procedure removing most of the nonspecific proteins bound to the surface. Figure 11(a) illustrates the time dependence of the differential reflectance corresponding to the stock antiserum diluted 140-fold in a 1-mg/ml BSA solution for the three classes of antigen coatings used in the experiments. Strong responses are observed for the KLH and DNP-BSA coatings, whereas negligible response is obtained for the BGG coated silver surface. The large changes for the DNP-BSA and KLH coated surfaces are expected since, as mentioned earlier, a DNP-KLH conjugate was used for immunization, thus producing antibodies to the DNP molecule as well as to the KLH binding sites. The larger variation for the DNP-BSA surface as compared to the IKLH surface may indicate either a higher concentration or a better antigen affinity of the DNP antibodies relative to the KLH antibodies in the serum samples. To confirm that the lack of response between the BGG coated Ag surface and the antiserum was indeed due to the absence of BGG antibodies in the mouse antiserum, an additional test was made with the BGG coated surface exposed to a 10 mg/ml of purified rabbit anti-BGG, diluted in a 1-mg/ml BSA solution. The result is shown as the upper curve in Fig. 11(b), with the lower curve giving the response to the mouse antiserum. As shown in Fig. 11(b), the BGG coated Ag surface reacts only with the anti-BGG solution. These results and the ones shown in Fig. 11(a) indicate the specificity of the method, i.e., antibody solutions will only react with the specific antigen coating on the Ag surface. This sequence of tests demonstrates that the SPI procedure is specific, and that even in a serum sample containing a large mixture of different proteins one can differentiate and measure a specific antibody. IV. Conclusions In this paper the SPS technique has been applied to the determination of specific antibodies in blood serum. Compared with the most sensitive labeling assays which are time-consuming and require somewhat sophisticated optical excitation and detection schemes, this approach has the following advantages: no labeling of reagents is required; a simple light source such as a He-Ne laser or an LED can be used for excitation; the detection system consisting of a pair of semiconductor photodetectors is small, lightweight and inexpensive; and rapid, sensitive, specific antibody concentration determinations are obtainable. In developing a SPI, one has to choose a metal surface that will provide the highest assay sensitivity, and silver is far superior to any other metal due to its inherent low optical absortion in the visible range. However, we have found that a single silver film deposited on a glass substrate5 6 is unstable when the metal surface contacts an aqueous medium. Under these conditions, it becomes difficult to differentiate reflectance changes due to antibody adsorption from insta1 November 1990 / Vol. 29, No. 31 / APPLIED OPTICS

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bility induced changes in the silver film, thus making the SPI unsuitable for most applications. However, by precoating the glass substrate with a chromium film, 30 A thick, prior to silver deposition, highly stable surfaces can be produced, making the approach amenable to the design of a practical immunosensor. The degree of surface stability achieved using this multilayer configuration was such that the protein films left on the surface after a given adsorption experiment could be removed with trichloroethane, followed by acetone and methanol rinsing. In these conditions, the surface state could be restored and the metal surface reused for additional experiments. During the course of this work, the adsorption of antigen proteins on the clean silver surface as well as desorption due to rinsing were investigated with the SPS approach. A simple theoretical model provides a linear relationship between the shift in the SP resonance angle and the effective protein thickness. By properly accounting for the molecular structure of the proteins involved, we were able to obtain excellent agreement between measured and predicted values for the effective protein thickness. The antigen adsorption studies also indicated that a 10% protein desorption takes place during rinsing, which may cause some nonspecific adsorption of serum proteins in the antibody measurements. This effect was eliminated in antibody adsorption experiments by blocking uncovered sites on the surface with a protein nonspecific to the protein reagents, such as BSA. Antibody concentration measurements from serum samples indicated that the SPI is capable of differentiating small amounts of specific antibodies in a much higher background of nonspecific serum proteins with high specificity. The detectability limit of the approach is comparable with the ones achievable by the RIA and ELISA methods. We would like to thank Hong Zhao from the Immunology Division of the Stanford University Hospital for providing and characterizing the antisera. E. Fontana is supported by a grant from Coordenadoria de Aperfeigoamento de Pessoal de Ensino Superior, Brazil. References 1. R. Edwards, Immunoassay: An Introduction (William Heinemann Medical Books, London 1985), p. 3..

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2. A. Voller and D. E. Bidwell, "Enzyme Immunoassays," in Alternative Immunoassays, W. P. Collins, Ed. (Wiley, New York, 1985), Chap. 6. 3. M. J. O'Sullivan, "Enzyme Immunoassay," in PracticalImmunoassay. The State of the Art, Wilfrid R. Butt, Ed. (Marcel Dekker, New York, 1984), Chap. 3. 4. A. Otto, "Spectroscopy of Surface Polaritons by Attenuated Total Reflection," in Optical Propertiesof Solids, New Developments, B. 0. Seraphin, Ed. (North-Holland, Amsterdam, 1975), Chap. 13. 5. B. Liedberg, C. Nylander, and I. Lundstrom, "Surface Plasmon Resonance for Gas Detection and Biosensing," Sens. Actuators 4, 299-304 (1983).

6. M. T. Flanagan and R. H. Pantell, "Surface Plasmon Resonance and Immunosensors," Electron. Lett. 20, 968-970 (1984). 7. E. Kretschmann, "Die Bestimmung optischer Konstanten von Metallen durch Anregung von Oberflachenplasmaschwingungen," Z. Phys. 241, 313-324 (1971). 8. H. Raether, "Surface Plasma Oscillations and their Applications," Phys. Thin Films 9, 145-261 (1977). 9. I. Pockrand, "Surface Plasma Oscillations at Silver Surfaces with Thin Transparent and Absorbing Coatings," Surf. Sci. 72, 577-588 (1978).

10. H. Raether, "Surface Plasmons and Roughness," in Surface Polaritons, V. M. Agranovich and D. L. Mills, Ed. (NorthHolland, Amsterdam, 1982), Chap. 9. 11. E. Fontana and R. H. Pantell, "Characterization of Multilayer Rough Surfaces by Use of Surface-Plasmon Spectroscopy," Phys. Rev. B 37, 3164-3182 (1988). 12. D. S. Campbell, "Mechanical Properties of Thin Films," in Handbook of Thin Film Technology, L. I. Maissel and R. Glang, Eds. (McGraw-Hill, New York, 1970), p. 12.30. 13. D. S. Campbell, Ref. 12, p. 12.9. 14. J. H. Weaver, C. Krafka, D. W. Lynch, and E. E. Coch, "Physics Data: Optical Properties of Metals Part. I (Fachinformationszentrum, FRG 1981) pp. 62-65. 15. D. Fasman, Ed., Handbook of Biochemistry and Molecular Biology: Proteins Vol III. (CRC Press, Cleveland 1976), pp. 372-380. 16. N. M. Senozam, J. Landrum, J. Bonaventura, and C. Bonaventura," Hemocyanin of the Giant Keyhold Limpet, Megathura crenulata," in: InvertebrateOxygenBindingProteins,J. Lamy and J. Lamy, Eds. (Marcel Dekker, New York, 1981), pp.703718.

17. C. Bonaventura and J. Bonaventura, "Respiratory Pigments: Structure and Function," in The Mollusca, P. W. Hochachka, Ed. (Academic, New York, 1983), Vol. 2, pp. 1-50. 18. B. Blomback and L. A. Hanson, Eds. PlasmaProteins, (Wiley, New York, 1976), p. 45. 19. I. Lundstrom, "Models of Protein Adsorption on Solid Surfaces," Prog. Colloid Polym. Sci. 82, 70-76 (1985).