Covalent coupling of immunoglobulin G to a poly(vinyl)alcohol-poly(acrylic acid) graft polymer as a...

ELSEVIER

Biosensors & Bioelectronics Vol. 13. No. 3-4, pp. 383-396, 1998 © 1998 Elsevier Science S.A. All rights reserved

Printed in Great Britain PII: S0956-5663(97)00114-0 0956-5663/98/$19.00

Covalent coupling of immunoglobulin G to a poly(vinyl)alcohol-poly(acrylic acid) graft polymer as a method for fabrzcattng the interfacial-recognztion layer of a surface plasmon resonance

immunosensor

Darren M. Disley, Jeff Blyth, David C. Cullen*, Hong-Xing Your, Saji Eapen¢ & Christopher R. Lowe

Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT, UK

(Received 2 June 1997; revised form received 25 September 1997; accepted 10 October 1997)

Abstract: The synthesis of a terminally thiolated poly(vinyl)alcohol (PVA) grafted with Poly (acrylic acid) (PAA) side chains is described. The PVA-PAA graft polymer (PVAg) was end-tethered to silver surfaces via the terminal thiol functionality and the resultant mobile, hydrophilic polymer matrix exploited for the covalent immobilization of large quantities of polyclonal goat (anti-hlgG) antibody (IgG) with low levels of non-specific adsorption. An SPR immunosensor, fabricated with an IgG-PVA-silver interfacial layer proved capable of performing a sensitive label-free assay of human IgG antigen (hlgG) with minimal non-specific binding interference. A detection limit (DL) for hlgG from serum of 0.8/xg/ml (5 nM) and an assay sensitivity of 0.66 ng hlgG/mm2/nM are reported. © 1998 Elsevier Science S.A. All rights reserved

Keywords: atomic force microscopy, covalent coupling, detection limit, immunoglobulin, immunosensor, interfacial-recognition layer, poly(acrylic acid), poly(vinyl)alcohol, surface plasmon resonance, sensitivity

*Current address, Cranfield Biotechnology Centre, Cranfieid University, Cranfield, Bedfordshire, MK43 0AL, UK tCurrent address, Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indi- anapolis, Indiana, 46202-525, USA ~Current address, Cambridge Sensors Ltd, Downhams House, Downhams Lane, Cambridge, CB4 1XT, UK

INTRODUCTION

Many studies of protein adsorption at the polymer-water interface have been carried out in the drive to develop suitable materials for: the fabrication of medical devices with anti-thrombogenic properties (Ishihara et al . , 1994; Hilborn, 1996; Keogh et al . , 1996; Litauzski et al . , 1997); the production of protein resistant contact lenses (Garrett and Milthorpe, 1996) and; the construction of high-affinity chromatographic supports

383

D. M. Disley et al. Biosensors & Bioelectronics

(Bottomly et al., 1995; Berna and Porath, 1996; Kubota et al., 1997), leading to a wealth of available information.

Protein adsorption to polymers is considered to be driven by a reduction in the free energy observed at the polymer-water interface, leading to entropically stable protein-surface interactions (Andrade, 1985). This would account for the general observation that hydrophilic polymers exhibit a stronger resistance to protein adsorption than do hydrophobic polymers (Rabinow et al., 1994; Andrade et al., 1996; Breton et al., 1996; Green et al., 1997).

The ability of hydrophilic polymer-coatings to resist protein adsorption has led to their employment as a means of covalently immobilizing immunoglobulins to the transducer region of optical (LOfas and Jonsson, 1990; Cush et al., 1993; Millot et al., 1995; Astles and Miller, 1994), piezoelectric (Nakanishi et al., 1996; Si et al., 1996) and electrochemical (Sadik and Vane- mort, 1996; Tiefenaur et al., 1997) immunosensors. Coating the sensor surface with a 3D hydrophilic polymer-matrix allows for controlled amounts of immunological-recognition component to be irreversibly immobilized with low levels of non-specific adsorption. Such a controlled immobilization can confer on the immunosensor; oper- ational stability, regenerability, assay sensitivity and analyte selectivity are characteristics not always exhibited by immunosensors employing non-polymer based immobilization methods (Muramatsu et al., 1987; Davies and Leafy, 1989; Zaitsev et al., 1991; Huber et al., 1992; Gizeli et al., 1992, 1993a, b).

The protocol first described by LOfas and Jons- son (1990) for a fast and simple covalent immobilization of immunoglobulins to a carboxymethyl- ated dextran (CMD) matrix, stands out as a means for fabricating the interfacial-recognition layer of immunosensors. This immobilization protocol forms the basis of the BIAcore (Jonsson et al., 1991) and Fisons IAsys (Cush et al., 1993) commercial biosensor technologies which have found wide application in immuno-specific interaction analysis (Jonsson et al., 1991; Buckle et al., 1993; Watts et al., 1994; Allauzen et al., 1995; Liedberg et al. , 1995; Gill et al. , 1996; Holwill et al., 1996; Neri et al., 1996; Hall and Winzor, 1997; Medina et al. , 1997; Myszka et al., 1997; Oddie et al. , 1997). Direct detection of target antigens from serum samples at medium concentration ranges (1-1000nM) have been reported,

384

with very low interference by NSB. The detection of target analytes at low concentrations ( < 1 nM) remains a problem due to the ratio of authentic signal to noise when only small amounts of ana- lyre are bound to the matrix. This is currently overcome for clinical applications by employing a sandwich-binding protocol to enhance the sensor response (Owen, 1997).

This paper describes the development of an alternative, generic, immunoglobulin immobilization surface for SPR immunosensors that is based on the employment of a terminally thiolated PVA grafted with PAA side chains (PVAg). The PVAg will spontaneously immobilize to silver SPR devices in a single 'mild' adsorption step and the resultant mobile, hydrophilic PVAg-modi- fled silver surfaces should allow the immobilization of large amounts of IgG antibody, via a carbodiimide coupling reaction, to be carried out with low levels of non-specific adsorption. SPR devices fabricated with such interfacial-recognition layers should be able to perform reagentless assays of hIgG antigen with a high degree of selectivity and sensitivity.

EXPERIMENTAL

Materials

Bovine serum albumin (BSA) was purchased from Sigma Immunochemicals, Poole, Dorset, UK. Coomassie Brilliant Blue G-250 solution, 1- ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride N-hydroxysulphosuccinimide, affinity-purified polyclonal goat (anti-hIgG) antibody, polyclonal hIgG antigen, human serum and Iodo-Beads T M were all purchased from Pierce Immunochemicals, Rockford, Illinois, USA. Mica sheets and chromium metal were obtained fr"bm Agar Scientific, Cambridge, UK. Acetic acid, acrylic acid monomer, ammonium cerium nitrate, 2,2-azoisobutyronitrile (AIBN), cetramide, dithiothreitol, dodecyl sulphate sodium salt (SDS), ethanol, ethanolamine, glutaraldehyde, glycine hydrochloride, ironmnitrate, heptane, mercap- toacetic acid, methanol, methyl ethyl ketone (MEK), 2-(N-morpholino)ethane sulphonic acid, nitric acid, pentane, poly(vinyl)acetate, poly(vinyl) alcohol, potassium ferricyanide, propan-2-ol, analytical grade silver shot, sodium dihydrogen orthophosphate, sodium iodide, sodium orthophosphate, sodium thiosulphate,

Biosensors & Bioelectronics Covalent coupling of immunoglobulin G

trichloroacetic acid, triethanolamine Triton X-100 and Tween 20 were all purchased from the Ald- rich chemical company, Gillingham, UK. Iodine isotope 125 [~25I] in carrier-free sodium iodide, was obtained from Amersham International plc, Aylesbury, UK. All solutions were made with high purity water (18 Ml2/cm) from an Elgastat water purification system at the Institute of Biotechnology, Cambridge, UK.

Methods

Synthesis of terminally thiolated pva Vinyl acetate (250g), previously distilled to remove inhibitors, was added to a three-necked, round bottom flask (500ml) and then placed under nitrogen with stirring. Mercaptoethanoic acid (1 ml) and 2,2-azoisobutyronitrile (AIBN, 0.1 g) in methanol (10ml) were added and the mixture refluxed for 50 min at 70°C. Further increments of mercaptoethanoic acid (1.5 ml) were added at hourly intervals, followed by a 5 h reflux of the reaction mixture, until a highly viscous reaction mixture was obtained. Heptane (800 ml), was used to remove unreacted vinyl acetate monomer and a further addition of heptane (200ml) was employed as a rinsing stage. Methyl(ethyl)ketone (MEK) (400 ml) was added, with stirring, until the crude acetylthiolated poly(vinyl)acetate (PVAc) went into solution. Pentane (800 ml) was added to precipitate the voluminous white acetylthiolated PVAc product which was then rinsed with further amounts of pentane (3 x 200 ml) and left under nitrogen for 48 h.

The methanolysis of the acetylthiolated PVAc was carried out by reaction, under nitrogen at 60°C, of 5 x 50 g portions of acetylthiolated PVAc with a NaOH (1.2 g) in methanol (100 ml) and MEK (60 ml) solution for 5 h. After leaving under nitrogen overnight, the reaction mixture was washed with methanol (2 x 150 ml), followed by several rinses in cold deionized water (100ml). The thiolated PVA (200g; 80% yield) was then dissolved in boiling water (100ml), cooled and stored in 25 ml aliquots under nitrogen.

Assay for thiol content of terminally thiolated pva Prior to acrylic acid grafting, the tbiol content of the modified PVA was determined by reaction of thiolated PVA (0.5 g), in deionized water (10 ml),

with an excess of standard iodine solution (0.1 M). Residual amounts of iodine were determined by titration with a standard sodium thiosulphate solution (0.1 M), which was repeated three times. The average amount of sodium thiosulphate consumed in the titrations was used to calculate the number of thiol groups incorporated per PVA molecule.

Grafting of acrylic acid monomers on terminally thiolated pva Thiolated PVA (13.5 g; 50/xmol) in degassed, deionized water (100ml) was added to a round bottom flask (250 ml) and placed under nitrogen. After rapid stirring for 5 min a small amount of potassium ferricyanide (7 nM) was added followed by further degassed, deionized water (25 ml). The reaction mixture was then stirred at room temperature for 3 h. Acrylic acid monomer was then added in excess to the reaction mixture (146/xM) and stirred for 5 min. The grafting reaction was initiated by the rapid injection of an amount of ammonium cerium nitrate (4.3 nM) in 0.01 M nitric acid (1 ml) under nitrogen. The reaction mixture was then stirred for 3 h at room temperature. The product of the graft polymerization was extracted with methanol (300 ml) and collected by rotor-centrifugation at 13 000 revolutions per min for 20 min. The PVAg (25g) was dried in an oven at 35°C for 24h and then dissolved in boiling water (100ml). Dithiothreitol (0.015 mM) was then added and the aqueous PVAg product cooled and stored in 20 ml aliquots under nitrogen.

Assay for carboxyl content of pvag The carboxyl content of the PVAg was determined using a method initially outlined by Helffer- ich (1962). The general procedure involved washing the PVAg (5 g) on a glass sinter with 2 M HC1 (50 ml) and then with nanopure water. The washed PVAg sample was then transferred to 0.1 M NaOH containing 0.5 M NaCI (100 ml) in a plastic bottle. The material stood for 24 h before an accurately measured sample of supematant (20 ml) was removed and placed into a conical flask (50ml) containing one drop of phenolphthalein solution (Basset et al., 1978). The pur- ple mixture was then back titrated with 0.1 M HC1 to a colourless end point. The volume of acid used was noted, before two further repetitions of the assay were performed. The material was washed with water (100ml), then acetone

385


(100 ml) and dried in an oven overnight at 60°C, before weighing to determine the dry weight of the material involved in the assay experiment. This procedure was also carried out on a sample of thiolated PVA as a control.

Assay for thiol content of pvag The thiol content of the PVAg was determined using a method described above, to see if the grafting procedure had any effect on the terminal thiol groups previously incorporated into the PVA. This protocol was performed before addition of dithiothreitol to the PVAg product and then again after addition.

Gel-permeation chromatography of pvau All gel permeation chromatographs were carried out by Rapra Technology Ltd, Shrewsbury, UK. Samples were run in dimethylsulphoxide with lithium bromide at 80°C on a PLgel mixed bed- B, 10/xm, 30cm GPC column (calibrated with dextran) at a flow rate of 1 ml/min. A refractive index detector was employed throughout and all results are expressed as 'dextran equivalent' molecular weights.

Cleaning of spr diffraction gratings and glass microscope slides An extensive cleaning and regeneration protocol was used throughout. The protocol consisted of: sonication for 5min at 25°C in ammonium cerium nitrate [1 M] followed by rinsing in nanopure water; soaking in Decon 90 soap solution 1% (w/v) for 5 min at 25°C; extensive rinsing and sonication in nanopure water; sonication for 10 min at 25°C in propan-2-ol, followed by over drying at 50°C for 20 min.

Deposition of silver films on spr diffraction gratings and glass microscope slides Silver films were deposited using a thermal evaporation technique. A chromium adhesion layer (2 nm) followed by a silver layer (100 nm) were deposited using an Edwards 306 A high vacuum metal evaporation unit at a pressure below 3 x 10 -7 Tort, a deposition rate of 0.5 nm/s and at a substrate temperature of 200°C.

Fabrication of silver-coated spr diffraction gratings and glass microscope slides with a pvag coating Silver-coated SPR diffraction gratings and glass microscope slides were incubated in a solution

386

of PVAg at concentrations of between 1-10% (w/v). The polymer was end-tethered to the silver surfaces via the formation of a covalent bond between the terminal thiol of the PVAg and the silver surface. Results are quoted as standard deviations from the mean of triplicate readings.

Static-contact angle measurements for pva u- modified surfaces The advancing static-contact angle of a single droplet of water placed at various locations on the PVAg-modified silver surfaces was determined using a Rame Hart Goniometer with the results being quoted as standard deviations from the mean of triplicate readings, unless otherwise stat- ed.

Non-specific protein adsorption to pvag-modified silver surfaces Unactivated PVAg-modified silver surfaces were incubated for 1 h at 25°C with aliquots (1 ml) of IgG (150/xg/ml) or pooled sheep serum in a 0-1 M sodium phosphate buffer at pH 6.5.

125I labelling of igg IgG was labelled according to the iodination method recommended by the reagent supplier (Bolton, 1985). The protein content of ~25I-labelled IgG was determined using the Coomassie dye binding method (Bradford, 1976). The radio- activity of the 125I-labelled IgG fractions were counted on an ISO-Data multi-head y counter.

Covalent coupling of igg to pvag-modified silver surfaces followed by radiolabelled experiments Silver-coated glass pieces (1 cm × 1 cm) previously fabricated with a PVAg layer were placed in a perspex immobilization block where a tight seal was made utilizing a Viton O-ring. The area available for covalent coupling was 46 mm 2. Aliquots (100 p,1) of an IgG antibody solution (0-150txg/ml), comprising known amounts of IgG and ~25I-labelled IgG in a pH 6.5 MES buffer were added to the immobilized silver pieces at 25°C. The carboxyl groups of the PVAg-modified silver surfaces were activated and antibody coupling was allowed to proceed for 1 h at room temperature in a 0.1 M 2-(N-morpholino)ethane sulphonic acid (MES) buffer, pH 6.5 containing 0.002 M of N-ethyl-N-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 0-005 M N-hydroxysulphosuccinimide (NHS). The silver pieces were then rinsed three times with aliquots


(100/xl) of the MES buffer to remove any loosely bound IgG antibody and then removed and counted on a ISO-Data multihead 500 3' counter. The total amount of coupled IgG was calculated and expressed as a mean surface loading of IgG.

Preferential coupling of J25I-labelled IgG compared to native IgG was checked for by adding aliquots (5 x 100/xl) of IgG antibody (150/xg/ml) containing an increasing amount of 125I-labelled IgG versus IgG (3160 cpm, 20 000 cpm, 31600cpm, 65000cpm and 100 000 cpm), to 1 cm x 1 cm silver coated glass pieces employing the same covalent coupling procedure described above. At the low IgG concentrations employed (0-150/xg/ml), it was found that no preferential adsorption occurs. A linear relationship between added ~25I-labelled IgG and covalently coupled ~25I-labelled IgG is observed even as the pro- portion of the total iodinated IgG is increased. This mirrors the observations made by other workers for ~25I-labelled IgG adsorption/coupling, at low concentrations, to silicon, gold and polymeric surfaces (Jonsson et al., 1991; Young et al., 1988; Lebedeva et al., 1991; Disley, 1995).

Antigen binding experiments followed by radiolabelled experiments Binding experiments were performed by the addition of aliquots (100/xl) of hIgG antigen- spiked human serum (0-50/xg/ml) comprising known amounts of hIgG and J25I-labelled hIgG to silver-coated glass pieces which had been previously immuno-sensitized with a covalently- coupled IgG-PVAg layer. The antibody-coated silver pieces were incubated with antigen at 25°C for 1 h before rinsing with aliquots [3 x 100/xl) of the experimentally-required buffer to remove any loosely bound antigen.

Non-specific binding determination followed by radiolabelled binding experiments Aliquots (1 ml) of hIgG antigen and of pooled sheep serum, pH 6.5 spiked with hIgG antigen (0-50/xg/ml) were added to silver-coated glass pieces, previously modified with PVAg and incubated with 1 ml aliquots of IgG antibody at an IgG concentration of 50/xg/ml, then incubated for 2 h with BSA (1% (w/v)). The binding medium employed was a 0.1 M sodium phosphate buffer, pH 6.5.

All radiolabelled experiments were performed in triplicate and results are quoted as standard deviations from the mean. The results are subject

to an additional _+ 5% error to that quoted, due to experimentally determined inefficiencies in the radioactive counting protocol employed.

Spr experiments All measurements were made using a SPR immunosensor incorporating a silver-coated diffraction grating, fabricated using a method described by Cullen et al. (1988). The device employed has been previously calibrated for IgG adsorption/binding using ~25I-labelled data obtained from experiments described in Disley (1995). All results shown are, as a consequence, also subject to an additional + 5% error margin to those quoted. SPR measurements were, in prac- tice, made by recording the intensity of the reflected angle at discrete values for quantitative determinations or as a function of time at fixed- incident angles for kinetic determinations. The resolution of the SPR device was 0.001 degrees. All equilibrium data were converted to immunoglobulin surface coverage values directly from external angular shifts and are therefore uncor- rected for cell contents. All SPR data are quoted as the standard deviations from the mean of triplicate readings.

Covalent coupling of igg to pvag-modified silver surfaces followed by spr PVAg-coated silver SPR devices were incubated for 1 h at room temperature with aliquots (1 ml) of IgG at IgG concentrations of 0-150/xg/ml, so as to electrostatically pre-concentrate the polymer matrix with IgG. The coupling medium employed was a 0.1 M MES buffer, pH 6.5. Quenching of excess surface carboxyl groups was achieved by passing a 1 M solution of ethanolamine hydrochloride (pH 8.5) over the IgG-modified silver surface.

Antigen binding experiments followed by spr Binding experiments were performed by the addition of 1 ml aliquots of hIgG and hIgG-spiked human serum (0-50/xg/ml), to silver-coated SPR devices which had previously been immuno-sensitized with a covalently-coupled IgG-PVAg layer. The antibody-coated-silver pieces were incubated with antigen at 25°C for 1 h before rinsing with aliquots (3 x 100/xl) of the experimentally- required buffer to remove loosely bound antigen.

387


Surface regeneration Dissociation of hlgG bound to lgG, covalently- coupled to PVAg-modified silver surfaces, was obtained by the washing of bound hIgG with a pulse of 0-001 M sodium phosphate-buffered glycine solution, pH 2.5 for 15 rain.

RESULTS AND DISCUSSION

Synthesis of pva containing a terminal thioi functionality

In the polymerization of vinyl acetate containing a terminal thiol (Fig. l(a)), the incremental addition of the thiol chain transfer reagent is necessary in order to obtain a polymer with a narrow molecular weight distribution. This is because the consumption of the reactive chain transfer agent, with a chain transfer constant (Dinaburg and Vansheidt, 1954) much larger than unity, is remarkably fast compared with that of the vinyl acetate monomer. This results in a decrease of the concentration of the former to the latter. The formation of PVAc having an acetylthiol group at one end was confirmed by GPC, where a mean 'dextran equivalent' molecular weight of 250 000 and a polydispersity ratio of 3 was obtained. These results are comparable to those reported in the literature for a similar synthesis (Sato and Okaya, 1993).

The methanolysis of acetylthiolated PVAc was carried out with sodium hydroxide in a nitrogen atmosphere. The mole ratio of sodium hydroxide to PVAc units was controlled to be approximately 1:1 and the degree of hydrolysis was found to be above 99%, indicating both acetyl groups of PVAc, as well as the acetylthiol end groups, had been cleaved. The presence of free thiol was obvious by a distinctive smell, typical of that given by thiol compounds.

The approximate number of thiol groups incorporated per PVA polymer chain was determined by iodometry to be 0.80, indicating that 80% of PVA chains contain a reactive thiol group. The uniformity of such thiol incorporation should allow for the end-tethering of PVA chains to silver surfaces via a spontaneous covalent-attachment.

(;rafting of acrylic acid monomers to terminal thiolated pva

The method used to produce grafted acrylic acid polymer chains on the thiolated PVA backbone

388

was initially described by Mino and Kaizerman (1958). In this method, acidic C e 4+ ions were used to initiate the grafting of acrylonitrile, acryl- amide and methyl acrylate monomers onto PVA. This method has since been used to produce a range of PVA-based materials for ion-exchange chromatography applications (Muller, 1986, 1990).

Because of the harsh conditions required to promote the grafting reaction, a protection of the terminal thiol groups was effected prior to the grafting reaction being performed. A controlled amount of potassium ferricyanide (7 nM) was added to the thiolated PVA to promote disulphide linkages between terminal thiol groups of the PVA molecules. It was thought that a degree of free thiol protection coupled to a stoichio- metricaly controlled addition of ammonium cerium nitrate in dilute nitric acid (4.3-6.4 nM thiol), would prevent further oxidation of terminal thiol groups to sulphoxide groups, whilst allowing the grafting of a number of acrylic acid side- chains. The disulphide bridges could then be reduced back to free thiol groups using dithiothreitol.

The grafting procedure (Fig. l(b)), involved adding an excess of acrylic acid monomer to a degassed solution containing a sample of thiolated PVA. Polymerization was initiated by adding a stoichiometric number of Ce 4+ ions dissolved in dilute nitric acid (4-3 nM). A reaction occurred quite quickly, detected by the disappearance of the orange reaction mixture colour as the Ce 4+ ions were reduced to Ce 3÷ ions. The orange colour faded to yellow over 30 rain, becoming colourless after 3 h when it was assumed the reaction had gone to completion.

The chemistry of polymer grafting onto PVA has been discussed in the literature (Odian and Kho, 1970). The chain growth radical polymerization is initiated by the abstraction of a hydrogen atom from PVA which is then oxidized to a hydrogen ion via a reversible ceric ion redox reaction (Mino and Kaizerman, 1958). The activated PVA polymers carry free radicals that rap- idly react with vinyl groups of the acrylic acid monomer in solution that initiate polymerization, exclusively on the PVA backbone, to produce 'tentacle type' grafted polymers containing carboxyl groups. An advantage of this procedure is that the density of hydroxyl groups on the PVA polymer, the initiator and the monomer concert-

Biosensors & Bioelectronics

(a)

'w"mCH2--CHe~Ac + CH3--~'---SH

Covalent coupling of immunoglobulin G

AIBN/Methanol _-

50min, 70°C OAc

+ VOAc incremental addition

l 5h, 50°C S (CH2--CH) n H

(b)

HS (CH 2

Methanol/MEK

( CH 2 --(~H)n H Na OH OAc 24 h, 60°C, N 2

K3Fe(CN )6 - - i H) n H 7nM, 3h, 25°C

OH

HS-- ( CH2--~3HI)n~H

- ( "S - - (C H 2 - - i H) n--~'2 H

OH

4.3 nM Ce4+/H ÷

- ( -S - - (CH 2 - - i H)n~2H 5 min~- OH

- ( ' -S-- (CH2 - -~ ) n--)~2 H

OH

CH 2 CHCO2H

OH

i + 0 ~ -i-

I

OH

Fig. 1. (a). Reaction sequence showing the synthesis of thiol-terminated PVA. (b). Reaction sequence showing the grafting of poly(acrylic acid) side-chains onto the carbon backbone of terminal-thiolated PVA.

trations can be controlled to determine the density and length of the grafted polymers.

The average molecular weight of the PVAg polymer, determined by GPC, was shown to be 460 000 'dextran equivalents'. The polydispersity ratio of 8 indicates that acrylic acid side chains of varying lengths are grafted to the PVA backbone. This deviates from the idealized structure of a PVAg polymer proposed by Pitfield (1992), which assumed a 100% conversion of acrylic acid

monomer and a 100% grafting yield, and pre- dicted to contain ten grafted acrylic acid side chains, of 100 monomers length, per PVA molecule. In this ideal structure, the ratio of acrylic acid monomers to PVA monomers in the final PVAg is 3:1. Considering the molecular weight of the thiolated PVA has only doubled following grafting, it can be presumed that~ a ratio of 1:1 is observed in this case.

The effect of the graft polymerization reaction

389


on the terminal thiol group of the PVA was determined by iodometry. The approximate total number of thiol groups per PVAg chain, after cleavage of the protective disulphide bridge with dithiothreitol, was determined to be 0.75, indicating that the stoichiometrically controlled grafting reaction had little effect on the terminal thiol group of the PVA.

The total number of carboxyl groups introduced into the thiolated PVA by the acrylic acid grafting reaction was determined by conversion of the anionic form of the PVAg to the protonated form by washing with HC1. Excess acid was then removed by washing with nanopure water before adding the polymer to 0.1 M NaOH containing 0.5 M NaC1. The H + ions were thus exchanged with Na + ions over 24h to ensure complete exchange had occurred. The H + ions released combine with free OH- ions to form water, resulting in a decrease in the pH of the soiution. Titration with 0-1 M HC1, using phenolphthalein as an indicator, neutralizes the remaining base. The difference in the volume of acid used, in comparison to a controlled sample, indicates the amount of H + ions released by the protonated carboxyl groups and from this the total carboxyl content of the PVAg polymer can be determined and thus the protein-binding capacity estimated.

The protein-binding capacity of 60 txM C O 0 / g polymer observed is approximately half the 113/xM COO-/g polymer reported by Pitfield (1992), for acrylic acid-grafted PVA- poly(tetrafluoroethylene)hexafluoropropylene (PVA-FEP) and is about the same as that observed for directly-synthesized carboxymethyl- ated PVA-FEP supports (56/xM COO-/g polymer). The reduced protein capacity is caused by the limited number of acrylic acid grafting sites that were initiated in order to protect the integrity of the terminal thiol functionality.

Characterization of pvag-coated silver surfaces

PVAg, from solution concentrations of between 1-10% (w/v), was end-tethered to silver surfaces via the formation of a covalent bond between the terminal thiol of the PVAg and the silver surface. The polymer surface loading, surface topography, surface hydrophilicity and the protein resistance of the PVAg-modified silver surfaces were all determined and a summary of the characteristics

390

of the PVAg-modified silver surfaces is shown in Table 1.

The presence of the PVAg on the silver surface was confirmed by SPR and indicated that when adsorbed from an optimum polymer solution concentration of 5% (w/v), a highly stable PVAg surface loading of 4.82 ng/mm 2 _+ 6% is obtained. Native PVA, adsorbed from solution to silver surfaces in control experiments, was found to be subject to large amounts of solvent-mediated desorption ( < 70%). This further indicates the stability given to the PVAg coating is due to a covalent-attachment of the terminal thiol of the PVAg to the bare silver surface. The polymer surface loadings observed are greater than the amounts observed by Jonsson et al. (1991) for a CMD covalently-attached to gold surfaces (3 ng/mm2), which indicates that an increased protein-capacity of the PVAg matrix may be obtainable compared to that observed for a CMD matrix.

Measurements of static contact-angles show that the hydrophilicity of the PVAg-modified silver surfaces (32 ° + 1 °) is greatly increased compared to that of both bare silver surfaces (114 ° + 1°), and of silver surfaces modifed with a self- assembled monolayer (SAM) of L-cysteine, 1,4- aminothiophenol, mercaptopropionic acid or thi- octic acid (64 ° - 94 ° + 1% Disley et al., 1997a). The hydrophilic nature of the PVAg-modified silver surfaces is supplemented by the hydroxyl groups that are omnipresent along the PVA backbone.

The resistance of PVAg-coated silver surfaces to protein adsorption (1.58 ng/mm 2 adsorbed _+ 4%) was found to be far greater than for silver surfaces modified with SAMs (3.5-6.8 ng/mm 2 adsorbed _+ 4%) (Disley et al., 1997a). This can be attributed to the increased hydrophilicity and mobility of the PVAg-modified silver surfaces compared to the comparatively rigid and hydrophobic SAM-modified silver surfaces. The mixed chain-length PVAg leads to a situation where a reduction in the freedom of movement of the highly-hydrophilic polymer chains, required for proteins to adsorb to the polymer-liquid interface, is energetically unfavourable due to entropy effects and leads to repulsion of protein adsorption.

Covalent coupling of igg to pvag-modified silver surfaces

IgG was electrostatically pre-concentrated on the negatively-charged PVAg-modified silver surfaces


TABLE 1 Summary of the characteristics of PVAg-modified silver surfaces.

PVAu concentration (% w/v)

PVA~ surface loading Mean static contact- (ng/mm 2) +_ 6% (3) angle (degrees)

IgG surface loading (ng/mm 2) + 4% (3)

Sheep serum surface loading (ng/mm 2) +

4% (3)

1 3.16 34 _+ 0.4 (3) 0.58 1.46 5 6.32 32 + 0.9 (3) 0.56 1.58

10 5-96 33 _+ 0.7 (3) 0.58 1-64

from a 0.1 M MES buffer, pH 6-5 (Jonsson et al., 1991). The charged groups of the PVAg matrix were then activated using an EDC/NHS protocol resulting in a rapid covalent coupling of the IgG to the PVAg. A pulse of 1 M ethanolamine hydrochloride at pH 8.5 was passed over the modified silver surfaces to deactivate any excess reactive ester groups. This pulse also had the effect of dislodging loosely-bound IgG.

SPR and radiolabelled studies demonstrated that reproducible IgG loadings of up to 38 ng/mm 2 ( _+ 5%) are obtained by electrostatic attraction concentration to the matrix, followed by EDC/NHS coupling. This implies an effective coating of up to six IgG layers and illustrates the relatively high protein binding capacity of the PVAg matrix. However, the surface loading is much lower than that obtained by Jonsson et al. (1991) for their CMD matrix (50 ng/mm2), inspite of the increased amounts of PVAg initially present on the silver surface.

A CMD matrix is known to have a highly flexible, uncrosslinked and open structure with a typical thickness of 100 nm (L/3fas and Jonsson, 1990). The lower protein-binding capacity observed for the PVAg matrix indicates a less flexible more closed structure. This almost cer- tainly inhibits the access of IgG to a number of the potential binding-sites on the activated matrix. It must be considered, however, that the PVAg is by no means optimized at this point and further investigations into the behaviour of the polymer in solution may generate knowledge that will allow for an opening of the polymer structure to be effected, thus allowing larger amounts of IgG to be immobilized.

D i r e c t h I g G a s s a y p e r f o r m e d b y a spr i m m u n o s e n s o r f a b r i c a t e d w i t h a P V A g - s i l v e r i n t e r f a c i a l l a y e r

For practical purposes, the amount of IgG immobilized to the PVAg matrix was controlled to be

approximately 20 ng/mm 2 + 5%. This was achieved by electrostatically pre-concentrating the PVAg matrix with a 100/xg/ml solution of IgG before chemical coupling. This control over the amount of immobilized IgG demonstrates the flexibility of the PVAg matrix. Such flexibility should enable the concentration of immobilized immunological-recognition components to be optimized for the various types of analyses to be performed.

Fig. 2 shows a typical set of binding curves for hIgG binding ( _+ 5%) to IgG covalently- coupled to a PVAg-modified silver SPR device. The total theoretical binding-site occupancy of hlgG at 50/xg/ml was determined to be 1.96 x 101° sites/mm 2 or 12%, decreasing to 1.83 × 101° sites/mm 2 or 11% when non-specific binding sites were blocked with BSA. This corresponds to an estimated non-specific binding level of 1.30 × 10 9 sites/mm 2 or 7% when expressed as a percent- age of the maximum available binding-sites on the PVAg matrix (1.6 × 10 ~ sites/mm2). These results were found to be within 8% of those obtained in identical radiolabelled experiments.

5 ,

~4,

m

r ~

o : .* : : ! *. l ! ! ! 5 10 15 20 25 30 35 40 45 50

hIgG Concentration (pg/ml)

Fig. 2. SPR binding isotherms showing hlgG binding to lgG antibody covalently-coupled to PVAg-modified silver surfaces from 0.1 M sodium phosphate buffer,

pH 6.5. [] = unblocked; 0 = BSA blocked.

391


The binding isotherm shown in Fig. 3, con- structed by subtracting the data from BSA- blocked binding isotherm from the unblocked binding isotherm (Fig. 2), represents specific hlgG binding to the IgG-PVAg-silver layer. From the truncated binding isotherm, the DL for hIgG from buffered samples was determined to be 0.1 /xg/ml or 0.67nM and the assay sensitivity to be 0.60 ng/mm2/nM.

The DL and sensitivity values demonstrate that the performance of an SPR immunosensor, fabricated with an IgG-PVAg immunological layer, for reagentless hIgG assay, is orders of magnitude greater than for one fabricated with a physically- adsorbed IgG layer (100nM: 0.04ng/mm2/nM) (Disley et al., 1997a) or a covalently-coupled IgG-SAM layer (26-50 nM: 0.09- 0.16ng/mm2/nM) (Disley et al., 1997a). The improved selectivity of the immunological layer is due to the large energy barrier to entropically- driven non-specific binding interactions that is exhibited by the mobile and hydrophilic PVAg matrix, whilst the increased assay sensitivity can be attributed to the large number of specific hIgG binding-sites that are generated by the large amounts of IgG immobilized in/on the PVAg matrix.

When performing a direct hIgG assay from serum samples, a higher background of SPR instrument noise, caused by non-specific binding of protein components of the serum sample to the IgG-PVAg layer, was observed. However, the background noise level, determined in radiolab-

elled studies to be 15%, is significantly reduced compared to those observed for physically- adsorbed IgG- (85%) or SAM-IgG (48-72%) immunological layers (Disley et al., 1997a). From the SPR binding isotherm shown in Fig. 4, the concentration at which hIgG can be detected above this noise level was determined to be 0.8 txg/ml or 5 nM and the assay sensitivity to be 0.66 ng/mm2/nM.

The performance of the chip is already an improvement on those obtained for most monolayer-based immunological coatings (Muramatsu et al., 1987; Davies and Leafy, 1989; Zaitsev et al., 1991; Gizeli et al., 1992; Lepesheva et al., 1994; Watts et al., 1994; Disley et al., 1997a). Low non-specific binding coupled to large protein binding capacity allows for reagentless detection of target analytes from buffered and serum samples in the low nM region (Table 2). With further work, the PVAg-silver sensor chip could provide a suitable alternative for performing label-free immunoassay to the CMD-coated gold (L6fas and Jonsson, 1990) and CMD-coated titania/hafnia sensor chips (Buckle et al., 1993).

The main advantage of the silver PVAg sensor chip is that fabrication of the silver sensor chip with the polymer matrix is performed in a single 'mild' adsorption step and does not, therefore, require the more complex fabrication protocol used by L0fas and Jonsson (1990).

A major disadvantage of employing the silver system, however, is the susceptibility of PVAg- modified silver sensor chips to damage by the

~ 0 . 6 ,

"• o.5,

0.4,

;~ 0.3,

~ 0.2.

r~ 0.1

}o

Fig. 3. SPR binding isotherm showing specific hlgG binding to IgG antibody covalently-coupled to PVA~,- modified silver surfaces from 0.1 M sodium phosphate buffer, pH 6.5. Isotherm was obtained by subtraction of the BSA blocked isotherm from the unblocked iso-

therm both shown in Fig. 2.

8

g s

3

2 r ~

. . . . . ." _. : : _" .~ 1

5 10 15 20 25 30 35 40 45 50 0 . ," ~ ," ; ~ ," ," ," ~ ,"

hIgG Concentrat ion (lag/ml) 0 5 10 15 20 25 30 35 40 45 50

hIgG Concentrat ion (pg/ml)

Fig. 4. SPR binding isotherm showing hlgG binding, above a background of non-specific binding, from 0.1 M sodium phosphate-buffered serum, pH 6.5 to lgG antibody covalently-coupled to PVA~-modified silver sur-

faces.

392


TABLE 2 A comparison of the capability of a SPR immunosensor, fabricated with an IgG (anti-hlgG) immunological layer, for performing hlgG assays from buffered samples. Figures in brackets denotes the DL for hlgG from

serum samples.

Analyte IgG immobilisation method Detection limit (nM) Reference + 5% (3)

hlgG Physical adsorption on silver 100 (167) Disley et al. (1997a) hlgG Thiol-mediated covalent-adsorption on silver 94 (127) Disley et al. (1997a) hlgG Covalent coupling to mercaptoproprionic acid monolayer 40 (58) Disley et al. (1997a)

on silver hlgG Site-directed coupling to 4-aminothiophenol-coated silver 26 (40) Disley et al. (1997a) hlgG Covalent coupling to PVAg-coated silver 0-67 (5)

low pH conditions required to regenerate the interfacial-recognition layer. At present only five cycles can be performed confidently before oxidation of the sensor chip occurs due to the low pH conditions required to regenerate the immuno- active surface. This compares to the 50 cycles that can currently be performed on a CMD-gold sensor chip (Liedberg et al., 1995). The PVAg coating could be employed with a gold SPR sensor chip which would exhibit a greater stability to the regeneration conditions employed. The use of a gold sensor chip would also enable the grafting reaction to be performed on the sensor chip itself, thus removing the limited number of PAA chains that can be grafted and potentially improving the protein capacity of the matrix.

CONCLUSIONS

A method for the rapid and facile covalent immobilization of IgG to PVAg-modified silver SPR devices has been developed. The silver sensor surface is covered with mobile 'tentacle like' PVAg chains (Pitfield, 1992) covalently-attached to the silver via a terminal thiol functionality (Finch, 1992; Sato and Okaya, 1993). The polymer layer covers the surface with hydroxyl groups which gives the surface a hydrophilic character.

This type of surface modification offers several distinct advantages for covalent immunoglobulin immobilization. First, the PVAg matrix provides a large immobilization capacity for immunological components, thus increasing the number of sub- sequently available analyte binding-sites. Sec- ondly, immobilization of immunoglobulins on a flexible PVAg chain provides a good degree of accessibility to target. Thirdly, the carboxyl

groups grafted on the PVA provide a chemical handle for the stable covalent immobilization of immunoglobulins. Finally, the hydrophilic and mobile nature of the PVAg chains provides an entropic barrier to non-specific protein interactions.

Ultimately, SPR immunosensors fabricated with a PVAg-coated silver or gold sensor surface, should offer the following features for concentration measurement of biological molecules compared with conventional immunoassay techniques: (a) methods can be developed and optimized to give real-time automated procedures for routine measurements; (b) concentration analyses may be performed on a kinetic rather than equilibrium basis, giving shorter assay times; (c) the method is readily extended to include techniques for enhancing the device sensitivity and selectivity; and (d) the sensor surface can be regenerated between measurement cycles.

ACKNOWLEDGEMENTS

The authors would like to thank the BBSRC for the funding of this work.

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