[acs symposium series] immunoanalysis of agrochemicals volume 586 (emerging technologies) ||...

Chapter 15

Liposome-Amplified Immunoanalysis for Pesticides

Stuart G. Reeves, Sui Ti A. Siebert, and Richard A. Durst

Analytical Chemistry Laboratories, Department of Food Science and Technology, Cornell University, Geneva, NY 14456—0462

Liposome-amplified competitive immunoassay systems for the herbicide alachlor, in both laboratory-flow injection and field immunomigration formats, are described. The preparation and characteristics of analyte-tagged liposomes, along with details of both types of assay are given. The laboratory assay is designed for automation and measurement of a large number of samples in the laboratory, whereas the field assay is designed for rapid field screening of large numbers of samples by non-technical personnel. The advantages of liposome immunoassay compared to more traditional formats are discussed, as are the problems posed by liposome curvature.

The use of immunoassays for pesticide monitoring is now well established, and a variety of laboratory and field assays have been developed (7,2). These immunoassays are mainly in the form of microtiter plate or tube ELISAs (enzyme-linked immunosorbent assays), where measurements are made of the color produced from a chromogenic substrate by the action of an enzyme conjugated to either an antibody or an analyte molecule. Such assays have been developed and are commercially available for the herbicide alachlor (3, 4), which is sometimes found as a contaminant of well water.

Previous studies (5-7) have demonstrated the advantages of liposome-encapsulated dye rather than enzymatically produced color to enhance the signal obtained in the competitive binding reaction of an immunoassay. In this communication the use of liposomes in a field assay format using immunomigration techniques and in an automated flow-injection immunoassay system is demonstrated, using alachlor as a model analyte. Liposomes provide instantaneous, rather than time-dependent, enhancement and offer considerable potential for both automated and field assays, for generic rather than specific assay reagents, and for multi-analyte assays.

0097-6156/95/0586-0210$12.00/0 © 1995 American Chemical Society

Dow

nloa

ded

by U

NIV

MA

SSA

CH

USE

TT

S A

MH

ER

ST o

n O

ctob

er 1

4, 2

012

| http

://pu

bs.a

cs.o

rg

Pub

licat

ion

Dat

e: M

arch

23,

199

5 | d

oi: 1

0.10

21/b

k-19

95-0

586.

ch01

5

In Immunoanalysis of Agrochemicals; Nelson, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

15. REEVES ET AL. Liposome-Amplified Immunoanalysis for Pesticides 211

The use of liposomes in immunoassay

Liposomes are lipid bilayer vesicles that are formed spontaneously when lipids are dispersed in water. During this formation they encapsulate a portion of the aqueous solution in which they were dispersed, and if this solution contains a marker molecule such as a dye, this will be present in the aqueous core of the liposome. Furthermore, if the analyte of interest is conjugated to a lipid, this can be incorporated into the liposome surface. A diagram of such liposomes is shown in Figure 1.

The principle of a competitive liposome immunoassay using such liposomes is shown in Figure 2. The tagged liposomes and the sample containing the analyte are passed over a solid surface to which antibody to the analyte of interest has been immobilized. Competition occurs between the free analyte molecules and the analyte molecules conjugated to the liposomes, and the number of liposomes that bind to the antibodies is inversely proportional to the amount of free analyte present. Unbound liposomes, which are directly proportional to the sample analyte, move out of the antibody region, and can be measured by an appropriate downstream detector. Alternatively, the bound liposomes can be measured in situ, or a detergent can be added in a flowing stream to release the marker which is then measured downstream.

The use of liposomes instead of the more usual enzyme-produced marker has several advantages. The lipid composition can be varied to give the liposomes different physical characteristics and almost any water-soluble marker can be encapsulated, giving rise to a broad range of possibilities for detection. The size of the liposome and the surface concentration of analyte tag can be varied and controlled accurately, giving more control over the experimental parameters of the assay. Enhancement is instantaneous, removing the requirement for a timed enzymatic incubation step, and the whole process lends itself to automation.

In this study, the widely used herbicide alachlor has been used as a model analyte. In order to raise antibodies, the analyte molecule was conjugated to a protein (BSA) (8), and to prepare alachlor-tagged liposomes it was conjugated to a lipid (DPPE - dipalmitoyl phosphatidyl ethanolamine) (9). In both cases available amino groups in the BSA and DPPE were thiolated and subsequently conjugated to the alachlor by nucleophilic displacement at the chloroacetamide group. This is shown in Figure 3, using conjugation to DPPE as the example.

Liposomes were then prepared by the reverse-phase evaporation method using a mixture of DPPC (dipalmitoyl phosphatidyl choline), cholesterol, DPPG (dipalmitoyl phosphatidyl glycerol), and alachlor-DPPE conjugate in a molar ratio of 5:5:0.5:0.01 (8-11). To improve their size distribution and homogeneity, the liposomes were passed through polycarbonate filters of 1.0 and 0.4 μπι. They were then purified by gel filtration and dialysis.

Characteristics

Stable liposome preparations have been made containing a variety of detectable compounds, but the work reported in this paper was carried out with 100 mM sulforhodamine Β as the encapsulant. In the case of the flow-injection assay the fluorescence was measured, whereas in the strip assay the high molar absorbance was utilized, and the color was estimated by eye or by densitometry.

These liposomes were stable for over 18 months when stored in the dark at 4 °C. They have also been stored for over 9 months at 25 °C and 6 months at 35 °C, again in the dark. Light was deleterious to membrane integrity, and caused dye leakage (or lysis) at a rate of 15-25% per month at room temperature. This is probably caused by energy dissipation effects from light absorbed by the dye inside the liposomes. This is supported by the fact that rapid lysis of the liposomes can be observed under the

Dow

nloa

ded

by U

NIV

MA

SSA

CH

USE

TT

S A

MH

ER

ST o

n O

ctob

er 1

4, 2

012

| http

://pu

bs.a

cs.o

rg

Pub

licat

ion

Dat

e: M

arch

23,

199

5 | d

oi: 1

0.10

21/b

k-19

95-0

586.

ch01

5


212 IMMUNOANALYSIS OF AGROCHEMICALS

Figure 1. The Structure of an Analyte-tagged Liposome

Figure 2. The Competitive-binding Reaction between Liposomes and Analyte Molecules for Antibody Sites on an Immunoreactor Solid Surface

Dow

nloa

ded

by U

NIV

MA

SSA

CH

USE

TT

S A

MH

ER

ST o

n O

ctob

er 1

4, 2

012

| http

://pu

bs.a

cs.o

rg

Pub

licat

ion

Dat

e: M

arch

23,

199

5 | d

oi: 1

0.10

21/b

k-19

95-0

586.

ch01

5


REEVES ET AK Liposome-Amplified Immunoanalysis for Pesticides

DPPE (NH2)

r SATA

NH 2 OH

Thlolated DPPE Alachlor

U P I D ^ ^ C ^ S H C l v J L / ^ o ^

j nor D P P E ^

DPPE-Alachlor Conjugate

Figure 3. Conjugation Reaction of Alachlor to DPPE

Dow

nloa

ded

by U

NIV

MA

SSA

CH

USE

TT

S A

MH

ER

ST o

n O

ctob

er 1

4, 2

012

| http

://pu

bs.a

cs.o

rg

Pub

licat

ion

Dat

e: M

arch

23,

199

5 | d

oi: 1

0.10

21/b

k-19

95-0

586.

ch01

5



microscope when the illumination is focused on the sample, and also by the observation that light-induced leakage does not occur in preparations where potassium ferrocyanide is the encapsulant (A. Edwards, unpublished data).

The diameters of the liposomes were measured by laser scattering in a LA-900 Particle Size Distribution Analyzer (Horiba Inc., Irvine, CA), using the manufacturers method, except that the usual sonication step was omitted to avoid lysis (rupture) of the liposomes (9). This gave an average diameter of 0.7 μπι. A second determination, using a different instrument (a Coulter LS 130), gave a diameter of 0.4 μπι. Using these figures, the concentration of DPPE-alachlor tag in the lipid mixture, and with published data for the cross-sectional area of lipids in membranes (72), certain parameters could be calculated (9) for both possible diameters. Experiments are underway to resolve this discrepancy, and figures calculated from both diameters are given in Table 1.

Flow-Injection Liposome ImmunoAssay (FILIA)

The majority of conventional immunoassays use the microtiter plate format. These plates are cumbersome to use in an automated assay, and furthermore, each individual well needs to be coated with antibody, leading to unavoidable well-to-well and plate-to-plate variations. These problems can be overcome by utilizing a flow-injection method with a reusable, regenerable immunoreactor column. The arrangement of the system in current use in our laboratory is shown in Figure 4 A. This system was modified from a previously published design (6).

The immunoreactor column contains an inert support, (in our case glass beads silanized with aminopropyl trimethoxy silane), with the antibody conjugated to it (anti-alachlor conjugated via glutaraldehyde in this study) (13). In a flow-injection system all samples pass through the same, reusable column, and calibration standards can be included in the runs to verify the accuracy of the results. It has been found possible to use such a column for weeks without any deterioration in performance. During this period there was no apparent leakage of antibody from the column, but after a certain period of time there would be a sharp decline in the response of the column. At this point the column packing material was replaced. Such a continuous flow system is very easy to automate.

In a single assay in such an apparatus, the sample and liposomes in the carrier stream are first passed through the column, where competitive binding occurs, with higher levels of sample analyte causing less liposomes to bind to the column. Unbound liposome and sample pass through the column and go to waste. The column is then washed to remove any non-specifically bound liposomes and other contaminants. Next, detergent is passed through to lyse the liposomes and release the marker, which is detected downstream and quantitated. Finally, carrier is once more passed through to regenerate the antibody by washing off bound analyte, presumably by a simple exchange mechanism driven by mass action effects, and then the column is ready for the next run. Because the regeneration was mild, there was no measurable loss of activity during this step. A typical trace from a single run is shown in Figure 5. This was based on alachlor-tagged liposomes containing sulforhodamine B, and utilizing fluorescence detection. The small initial peak (A) is from the quenched fluorescence of intact liposomes that do not bind to the column, with the second, larger peak (B) produced by the released dye being the one that is used for quantitation. With such a system it is possible to quantitate alachlor down to the 10 ppb level, as shown in Figure 6, with a total assay time of 5 minutes. This level of detection is not as low as that obtained by a microtiter plate ELISA (0.1 ppb), and also higher than the MCL (Minimum Contaminant Level) for alachlor as defined by the EPA.

Dow

nloa

ded

by U

NIV

MA

SSA

CH

USE

TT

S A

MH

ER

ST o

n O

ctob

er 1

4, 2

012

| http

://pu

bs.a

cs.o

rg

Pub

licat

ion

Dat

e: M

arch

23,

199

5 | d

oi: 1

0.10

21/b

k-19

95-0

586.

ch01

5



A. Current Design

Carrier

Detergent

Sample

\

- K l Valve

Immunoreactor Column

^Detector^

B. Future Design

Sample

Carrier ·

Detergent < Valve

Immunoreactor Column

Reagent A -Valve

^Detector^

Figure 4. Schematic Diagram of the How-Injection System A. The current FILIA design B. A potential future design, using post-column addition and a mixing/holding coil.

Dow

nloa

ded

by U

NIV

MA

SSA

CH

USE

TT

S A

MH

ER

ST o

n O

ctob

er 1

4, 2

012

| http

://pu

bs.a

cs.o

rg

Pub

licat

ion

Dat

e: M

arch

23,

199

5 | d

oi: 1

0.10

21/b

k-19

95-0

586.

ch01

5



30 π

Figure 5.

1 2 3 4 5

Time (min)

Trace of a Typical FELLA Run

- 3

M 3 •Sa

10H

10 100 Alachlor (ppb)

1000

Figure 6. Alachlor Dose-Response Curve, FILIA System

Dow

nloa

ded

by U

NIV

MA

SSA

CH

USE

TT

S A

MH

ER

ST o

n O

ctob

er 1

4, 2

012

| http

://pu

bs.a

cs.o

rg

Pub

licat

ion

Dat

e: M

arch

23,

199

5 | d

oi: 1

0.10

21/b

k-19

95-0

586.

ch01

5


15. REEVES ET AL. Liposome-Amplified Immunoanalysis for Pesticides in

Various factors can be manipulated to alter the characteristics of the assay. Studies are currently underway to examine the effects of flow rate, column-bound antibody concentration, liposome size and surface concentration of the analyte tag on the assay. The object is to produce an assay with the required sensitivity which takes the minimum possible time, so that a large number of assays can be carried out each day. Recent experiments (G. Rule, unpublished data) suggest that the required limit can be reached under the correct conditions. Furthermore, other possible detection methods are being studied, e.g. chemiluminescence, and this would require addition of other reagents after the competition step. The design of the FILIA system allows such additions, as is common in flow-injection systems, and an example of how this could be arranged is shown in Figure 4B.

The exact amount of amplification due to the liposomes is currently unknown. If one liposome bound to one antibody, then it would be in the order of one million, the number of molecules entrapped per liposome (Table 1). However, multiple binding and shadowing (see later) reduce this considerably. At this point there is no fluorescent analog of alachlor available, so this control cannot be run. However, such an analog is currently being synthesized, and this will allow these measurements to be made.

Field immunomigration strip assay

An extra-laboratory assay for alachlor has been developed, using immunomigration techniques (9). A liposome and alachlor mixture is allowed to migrate up a strip of absorbent material on which anti-alachlor and egg white avidin zones have been immobilized. The competition occurs in the antibody zone, and the amount of liposome bound (quantified as the amount of color from encapsulated sulforhodamine B) is inversely proportional to the amount of analyte in the solution. A second (capture) zone containing egg white avidin to bind the liposomes, collects all of the liposomes that do not bind to the antibody zone, and thus the amount of color in this zone is directly proportional the amount of analyte in the sample. Quantitation in either zone is possible, with the use of the second zone being more intuitive and preferred. This assay has the potential for rapid field screening of analytes of interest, and is simple enough to be used by non-technical personnel.

In this assay, a protein-binding membrane with a plastic backing to provide rigidity was required, and porous nitrocellulose membranes (> 3μπι pore size) supported in this manner were found to be the most suitable. An automatic thin-layer plate sample applicator (Camag Linomat IV) was used to dispense the antibody and egg white avidin solutions onto spatially separate and well-defined zones of the membrane for immobilization. The membrane sheet was 8.0 cm high and 15.5 cm wide for later subdivision into strips 5 mm in width. The protein-coated membrane was blocked with 2% PVPP (polyvinylpyrrolidone) and 0.002% Tween-20 in TBS (Tris buffered saline) to prevent non-specific binding. The prepared sheets were vacuum dried and stored at 4°C in the presence of silica gel desiccant until ready for use. They were cut into 5 mm wide strips using a paper cutter when needed. The final strips had a 5 mm long antibody zone 15 mm above the bottom of the strip and a similar egg white avidin zone 15 mm above the antibody zone.

The prototype assay consists of a reagent containing alachlor-tagged liposomes and sample, and a test strip as described above. The assay is performed by dispensing 2 drops of the sample or control solution and 1 drop of a three-times concentrated buffer into a 10 χ 75 mm glass test tube, mixing the contents, and adding 1 drop of a liposome solution. The test tube is shaken mildly to mix the contents and the test strip is inserted into the tube; the strip is left in the tube until the solution front reaches the end of the strip (about 9 min); the strip is removed and air dried. The color intensity of

Dow

nloa

ded

by U

NIV

MA

SSA

CH

USE

TT

S A

MH

ER

ST o

n O

ctob

er 1

4, 2

012

| http

://pu

bs.a

cs.o

rg

Pub

licat

ion

Dat

e: M

arch

23,

199

5 | d

oi: 1

0.10

21/b

k-19

95-0

586.

ch01

5



Table 1. Liposome Characteristics

All parameters were calculated using an analyte-tag concentration of 0.1%, and one of the two measured values for diameter. The sulphorhodamine Β concentration in the liposomes was assumed to be the same as that used in the encapsulating procedure, and the lamellarity was assumed to be 1.

Mean Diameter 0.7 μπι 0.4 μηι Volume of Each Liposome 1 8 χ 1Q-10 μ 1 ^ 3 4 χ 1Q-11 ^ Liposome Concentration 1.2 χ 108 per 6.0 χ 108 per μΐ. SRB (Concentration) 100 mM 100 mM SRB (molecules per liposome) 9 6x 10̂ 1 8 χ 10̂ Alachlor (molecules per liposome) 3 5x10^ l l x l 0̂

the antibody zone and the egg white avidin capture zone are estimated either visually or by scanning densitometry.

Figure 7 shows diagrammatically the results obtained with a series of alachlor standards (A=0, increasing B<C<D), with the decrease in color of the antibody zone with increasing concentrations of added alachlor, and the concomitant increase in the color of the capture zone.

Dose-response data obtained by scanning densitometry of strips run in the presence of various concentrations of alachlor are shown in Figure 8. The response in both the antibody and avidin zones varied logarithmically with alachlor concentration, and both were estimated to be able to detect 5-10 ppb alachlor. When these strips were assessed visually, a similar determination could be made, but at low levels of added alachlor it was somewhat easier to detect increases of red color over a white control (capture zone) than decreases in color intensity (antibody zone). There are still difficulties with the reproducibility of the strips. These seem to be due to heterogeneity in the supplied sheets of plastic-backed nitrocellulose, and this problem is currently being addressed. As in the case of the FILIA, the detection limit is not as low as is required, but this can be improved down to the 1 ppb level by manipulation of various parameters (T. Siebert, unpublished data).

Figure 9 is a diagram of how the prototype strip could be developed into a user-friendly field device, using visual estimation of the color by comparison with a color card, or by measurement in a calibrated reflectometer (14). It both cases it is anticipated that the capture zone would be used for measurement rather than the antibody zone. A further possibility is for the production of multianalyte strips. A series of antibody zones, each to a different analyte, could be applied to the strip, and a suitable mixture of liposomes applied.

The effect of liposome size and analyte-tag density

In any liposome immunoassay system, the analyte density (concentration) on the surface of the liposome will have an obvious effect on the binding and competitive reactions. What is less immediately obvious is that liposome size can also have an effect.

A scale drawing of the surface view of a liposome with the concentration of analyte tag that has been routinely used, along with scale drawings of antibodies, is shown in Figure 10 A. Assuming random distribution of the tag molecules on the

Dow

nloa

ded

by U

NIV

MA

SSA

CH

USE

TT

S A

MH

ER

ST o

n O

ctob

er 1

4, 2

012

| http

://pu

bs.a

cs.o

rg

Pub

licat

ion

Dat

e: M

arch

23,

199

5 | d

oi: 1

0.10

21/b

k-19

95-0

586.

ch01

5



} Capture /Measurement 7MII>

} Immobilized Antibody Zone

A B C D

Figure 7. Diagram of a Series of Strip Assays

Figure 8.

Antibody Zone

Avidin Zone

10 100 1000

Alachlor (ppb)

Alachlor Dose-Response Curve, Strip Format

Dow

nloa

ded

by U

NIV

MA

SSA

CH

USE

TT

S A

MH

ER

ST o

n O

ctob

er 1

4, 2

012

| http

://pu

bs.a

cs.o

rg

Pub

licat

ion

Dat

e: M

arch

23,

199

5 | d

oi: 1

0.10

21/b

k-19

95-0

586.

ch01

5



Sample, liposome and buffer solutions

Color intensity measurement

End-point indicator

Air vent

Τ τ η π η ι ι η η τ τ Π grntlrvn strip

Antibody Zone

Capture Zone

Figure 9. Diagram of Proposed Strip Cassette

Figure 10. Diagram of Liposome Surface and Analyte/Antibody Interaction (Drawn to Scale)

Dow

nloa

ded

by U

NIV

MA

SSA

CH

USE

TT

S A

MH

ER

ST o

n O

ctob

er 1

4, 2

012

| http

://pu

bs.a

cs.o

rg

Pub

licat

ion

Dat

e: M

arch

23,

199

5 | d

oi: 1

0.10

21/b

k-19

95-0

586.

ch01

5



liposome surface, then the spacing is such that decreasing the spacing between analyte molecules (i.e. increasing the surface concentration) will presumably increase the number of binding interactions between the antibody and analyte tag on each individual liposome until the liposomes form a monolayer and no more can be packed on the surface. The number of alachlor-tag to antibody interactions per liposome will logically affect the number of free molecules required to "out compete" a liposome, and thus should affect the sensitivity of the assay. However, this diagram does not take into account the curvature and deformability of the liposome, or the mobility of DPPE-alachlor in the bilayer.

If we consider the immunoreactive surface, in either the FELLA or strip assay, to which the antibody is bound as a flat surface, then scale drawings of cross-sections can be made, and these are shown in Figure 10 Β. If one assumes that there is no deformation of the liposomes (certainly not the case), then as the diameter decreases, the chances of multiple interactions of a single liposome with several antibodies decreases.

These spatial effects, along with the fact that the system, like many flow-injection systems, is not at equilibrium, must be taken into consideration when attempting to modify and develop both the immunomigration and FELLA assays.

Acknowledgments

This work was supported in part by the Cornell Biotechnology Program, the National Institute of Environmental Health Sciences, National Institutes of Health, DHHS, under the Superfund Research and Education grant no. 1 P42 ES05950-01, and by the CSRS/USDA Water Quality Special Grants Program #93-34214-8846. Any opinions, findings, conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the Cornell Biotechnology Program, the U.S. Dept. of Health and Human Services or the U.S. Dept. of Agriculture.

References

1 Kaufman, Β. M.; Clower, M. Immunoassay of pesticides. J. Assoc. Off. Anal. Chem. 1991, 74, 239-247.

2 Immunochemical Methods for Environmental Analysis. (1990) Van Emon, J. M. and Mumma, R. O., eds, ACS, Washington, DC.

3 Feng, P. C., Wratten, S. J., Horton, S. R., Sharp, C. R. and Logusch, E. W. (1990) Development of an Enzyme-Linked Immunosorbent Assay for Alachlor and Its Application to the Analysis of Environmental Water Samples. J. Agric. Food Chem. 38, 159-163.

4 Lawruk, T. S., Hottenstein, C. S., Herzog, D. Ρ and Rubio, F. M. (1992) Quantification of Alachlor in well water by a novel magnetic-particle based ELISA. JBull. Environ. Contm. Toxicol 48, 643-650.

5 Locascio-Brown, L., Plant, A. L., Horvath, V. and Durst, R. A. (1990) Liposome Flow Injection Immunoassay: Implications for Sensitivity, Dynamic Range, and Antibody Regeneration. Anal. Chem. 62, 2587-2593.

6 Durst, R. Α., Locascio-Brown, L. and Plant, A. L. (1990) Automated Liposome-Based Flow Injection Immunoassay System. In "Flow Injection Analysis Based on Enzymes or Antibodies", R.D. Schmid, ed., GBF Monograph Series, 14, VCH Publishers, Weinheim, FRG, 181-190.

7 Yap, W. T., Locascio-Brown, L., Plant, A. L., Choquette, S. J., Horvath, V., and Durst, R. A. (1991) Liposome Flow Injection Immunoassay: Model Calculations of Competitive Immunoreactions Involving Univalent and Multivalent Ligands, Anal. Chem. 63, 2007-2011.

Dow

nloa

ded

by U

NIV

MA

SSA

CH

USE

TT

S A

MH

ER

ST o

n O

ctob

er 1

4, 2

012

| http

://pu

bs.a

cs.o

rg

Pub

licat

ion

Dat

e: M

arch

23,

199

5 | d

oi: 1

0.10

21/b

k-19

95-0

586.

ch01

5


222 I M M U N O A N A L Y S I S OF A G R O C H E M I C A L S

8 Reeves S. G., Roberts, Μ. Α., Siebert, S. T. A. and Durst, R. A. (1993) Investigation of a Novel Microtiter Plate Support Material and Scanner Quantitation of Proteins, Phospholipids and Immunoassays. Anal Letters, 26, 1461-1476.

9 Siebert, S. Τ. Α., Reeves, S. G. and Durst, R. A. (1993) Liposome Immunomigration Field Assay Device for Alachlor Determination. Anal. Chim. Acta, 282, 297-305.

10 Szoka, S., Olsen, F., Heath, T., Vail, W., Mayhew, E. and Papahadjopoulos, D. (1980) Preparation of Unilamellar Liposomes of Intermediate Size (0.1-0.2

µm) by a Combination of Reverse Phase Evaporation and Extrusion Through Polycarbonate Membranes. Biochim. Biophys. Acta, 601, 559-571.

11 O'Connell, J. P., Campbell, R. L., Fleming, B. M., Mercolino, T. J., Johnson, M. D. and McLaurin, D. A. (1985) A Highly Sensitive Immunoassay System Involving Antibody Coated Tubes and Liposome Entrapped Dye. Clin. Chem. 31, 1424-1426.

12 Israelachvili, J. N. and Mitchell, D. J. (1975) A Model for the Packing of Lipids in Bilayer Membranes. Biochim. Biophys. Acta, 389, 13-19.

13 Reeves, S. G., Rule, G. S., Roberts, Μ. Α., Edwards, A. J., Siebert, S. T. A. and Durst, R. A. (in press) Flow-Injection Liposome ImmunoAnalysis (FILIA) for Alachlor. Talanta.

14 Durst, R. Α., Siebert, S. T. A. and Reeves, S. G. (1993) Immunosensor for Extra-Lab Measurements based on Liposome Amplification and Capillary Migration. Biosen. Bioelectron. 8, xiii-xv.

RECEIVED November 17, 1994

Dow

nloa

ded

by U

NIV

MA

SSA

CH

USE

TT

S A

MH

ER

ST o

n O

ctob

er 1

4, 2

012

| http

://pu

bs.a

cs.o

rg

Pub

licat

ion

Dat

e: M

arch

23,

199

5 | d

oi: 1

0.10

21/b

k-19

95-0

586.

ch01

5


[acs symposium series] immunoanalysis of agrochemicals volume 586 (emerging technologies) ||...

Documents