WO2003052423A1

WO2003052423A1 - Method of preparation of a biosensing surface and biosensing surface

Info

Publication number: WO2003052423A1
Application number: PCT/FI2002/001040
Authority: WO
Inventors: Jouko Peltonen; Petri Ihalainen
Original assignee: Jouko Peltonen; Petri Ihalainen
Priority date: 2001-12-18
Filing date: 2002-12-18
Publication date: 2003-06-26
Also published as: AU2002352292A1; FI20012495A0

Abstract

The present invention relates to a method of preparation of a biosensing surface where a host matrix comprising at least one analyte specific binding agent and at least one analytically inert matrix agent comprising binding units is dissolved to form a host matrix solution. The host matrix solution is applied onto a subphase surface, where it forms an oriented monolayer, which is compressed. A solid metal substrate is brought in contact with the monolayer on the subphase surface, and the monolayer is transferred onto the solid substrate by using hydrophobic interactions between the monolayer and the solid substrate. The monolayer is also bound covalently to the solid substrate via binding units comprised in the analytically inert matrix agent molecules of the host matrix. The invention relates also to a biosensing surface.

Description

METHOD OF PREPARATION OF A BIOSENSING SURFACE AND BIOSENSING SURFACE

FIELD OF THE INVENTION

The present invention relates to preparation method of a biosensing surface defined in the preambles of the independent claims presented hereafter. Furthermore, the invention relates to a biosensing surface.

BACKGROUND OF THE INVENTION

There is an increasing demand for simple, selective, sensitive and easy-to-use biosensors for the sensing of various diagnostic immunoreactions. A biosensor usually comprises a molecular recognition site, i.e. receptor, and a transducer, which transforms a biochemical reaction to a measurable signal. A method to produce biosensing surfaces is Langmuir-Blodgett technique, with which solid- supported highly organised organic thin films can be produced.

The Langmuir-Blodgett film-forming technique has some differences compared to other film-forming techniques, such as self-assembly (SA). Self-assembled monolayers are formed spontaneously without external intervention by immersion of a substrate into a solution containing amphiphilic molecules. Molecules are absorbed and spontaneously organised on the substrate surface. Monolayers formed by self-assembly have usually a varying degree of molecular orientation and order. Accordingly defects and distortions in the monolayer uniformity are included [Hari Singh Nalwa (ed.)].

Even if SA films are simple and fast to produce, in biosensor applications they have a disadvantage of frequently showing high non-specific protein binding. On the other hand, SA films showing low non-specific binding require a long and complicated protocol of production [Hari Singh Nalwa (ed.)]. For mixed SA films the problem is the non-exact state and kinetic instability of mixing of the film components. The risk for phase segregation is also obvious [Allara, Folkers et al.]. Therefore, the relatively low degree of organization and functionality of SA films renders them generally inferior to Langmuir-Blodgett films in biosensor applications.

The Langmuir-Blodgett (LB) film-forming technique allows the preparation of monomolecular layers of controlled density on a solid substrate. The LB technique is based on letting a film-forming surface active compound orient itself at the interface between two different phases, for example at the interface between a liquid and a gas. A surface active compound dissolved in a suitable organic solvent or solvent mixture is spread onto the surface of a liquid, i.e. subphase, contained in a trough. The hydrophilic part of the molecule orients itself towards the liquid, and the hydrophobic part, i.e. the lipophilic part, in a direction away from the liquid. From the film spread as a monomolecular layer, i.e. Langmuir monolayer the solvent evaporates rapidly. By means of a barrier the available surface area of the liquid-gas-interface can be controlled, the total area of and hence the molecular density within the Langmuir monolayer thus decreasing or increasing. By means of the barrier it is thus possible to regulate the surface tension of the film, which is inversely proportional to the surface pressure of the film, and which is determined by measuring the force exerted on a sensor in the film by means of a sensitive balance.

When a support, which forms the substrate, is transported through the interface, preferably at constant speed, the film is deposited as a monomolecular layer onto the substrate, the lipophilic part towards the substrate when the support is transported through the interface in a direction from air to water, and the hydrophilic part towards the substrate when the support is transported through the film in the opposite direction. The thickness of the individual LB film layer is dependent on the organic compound used for the preparation of the film and especially on the length of the aliphatic chains contained therein.

The Langmuir monolayer may be transported onto a solid substrate also by using a horizontal deposition method. In this so-called Langmuir-Schaefer (LS) technique the substrate is brought in contact with the Langmuir monolayer floating on the subphase so that the substrate is oriented parallel, to the monolayer plane. The Langmuir monolayer becomes physisorbed to the substrate and remains on its surface when the substrate is lifted off or pushed through the interface. The LS technique is a faster and easier deposition method compared to the LB technique.

Monolayers prepared by LB and LS techniques are smooth and even with a high molecular orientation. High-density monolayers are obtained as the molecules in the monolayer can be packed in the closest possible arrangements allowed by the size of the head and/or tail groups. However, it has been difficult to use LB and LS monolayers in practical applications, as the monolayer film is only weakly physisorbed on the solid support, which has led to poor mechanical, chemical and thermal stability of monolayers, especially of films deposited onto hydrophobic surfaces. Many different solutions have been suggested over the years, e.g. use of preformed polymers, reactive amphiphiles polymerisable at the subphase/gas interface, or deposition of multilayers instead of monolayers [Rolandi, Bodalia and Duran, Arslanov, Laschewsky et al.].

SUMMARY OF THE INVENTION

The object of the present invention is, among other things, to solve or minimise the problems and disadvantages existing in the prior art.

Another object of the invention is to provide a simple method of preparation of mechanically, chemically and/or thermally stable biosensing surfaces. A third object of the invention is to provide a mechanically, chemically and/or thermally stable biosensing surface.

In order to achieve the above-mentioned objects the present invention is characterised in what is defined in the independent claims presented hereafter.

A typical method of preparation of a biosensing surface according to present invention comprises the following steps: Method of preparation of a biosensing surface comprising the following steps:

- dissolving a host matrix comprising at least one analyte specific binding agent and at least one analytically inert matrix agent comprising binding units to form a host matrix solution,

- applying the host matrix solution onto a subphase surface, where it forms an oriented monolayer,

- compressing the monolayer,

- bringing a solid metal substrate in contact with the monolayer^'on the subphase surface, and

- transferring the monolayer onto the solid substrate by using hydrophobic interactions between the monolayer and the solid substrate,

- binding the monolayer also covalently to the solid substrate via binding units comprised in the analytically inert matrix agent molecules of the host matrix.

A typical biosensing surface according to the present invention comprises - a solid metal substrate coated on a support structure,

- an oriented host matrix monolayer on the solid substrate,

- comprising molecules of at least one analyte specific binding agent and at least one analytically inert matrix agent, and

- bound covalently to the solid substrate via binding units comprised in the molecules of the analytically inert matrix agent of the host matrix. BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further illustrated in the following non-limiting drawings and figures.

Figure 1 The synthetic route for the preparation of DSDPPC and the molecular structure of DPPGL,

Figure 2 A simplified schematic illustration of deposition of monomolecular layer onto a substrate surface according to one embodiment of the present invention,

Figure 3 Pressure-area isotherms of DSDPPC on a pure water subphase at different temperatures,

Figure 4 Pressure-area and surface potential isotherms of pure DSDPPC (A), DPPC (B) and DPPGL (C) monolayers on a pure water subphase at 20.0 ± 0.4 °C,

Figure 5 Pressure-area isotherms of DSDPPC/DPPGL mixed monolayers on a pure water subphase at 20.0 + 0.4 °C.

Figure 6 Mean molecular area for the mixed monolayer of DSDPPC/DPPGL as a function of the molar fraction of DSDPPC at 20.0 ± 0.4 °C. The dotted lines in the figure demonstrate the deviation of the data points from the linear dependence of molecular area on the mixing ratio of the film components. Figure 7 Height images of LS monolayers of pure DSDPPC on a gold substrate as a function of exposition time. Gold substrates were kept on the subphase surface at surface pressure 30 mN/m for 20 s (Fig. 7B), 1 min (Fig. 7C), 3 min (Fig. 7D), 15 min (Fig. 7E) and 2 h (Fig. 7F). Also shown is typical height image of a pure gold substrate (Fig. 7A). The height scale is 5 nm, and the image size is lxl μm .

Figure 8 R_rms (•) and the contact angle (o) values of LS monolayers of pure DSDPPC on a gold substrate as a function of exposition time.

Figure 9 Height images of binary DSDPPC/DPPGL monolayers on gold for the mixing ratios of 95:5 (Fig. 9A) and 80:20 mol-% (Fig. 9B). Gold substrates were kept on the subphase surface at a surface pressure 30 mN/m for 2 hours. The height scale is 10 nm, and the image size is 2x2 μm².

Figure 10 Height images of Fab' fragment (Fig. 10A and 10B), BSA (Fig. 10C and Fig. 10D) and hlgG-exposed (Fig. 10E and Fig. 10F) DSDPPC/DPPGL monolayers on gold for the mixing ratios of 95:5 (Fig. 10A, Fig. 10C and Fig. 10E) and 80:20 mol-% (Fig. 10B, Fig.

10D and Fig. 10F). Both monolayers were horizontally deposited from Langmuir film at a surface pressure of 30 mN/m, the deposition time being 2 hours. The height scale is 10 nm, and the image size is 2x2 μm

DETAILED DESCRIPTION OF THE INVENTION

The term "host matrix", when used in context of the present invention, comprises the whole matrix, which forms the monomolecular layer, i.e. monomolecular film, used as a biosensing surface. Host matrix encompasses both molecules of the analyte specific binding agent and molecules of analytically inert matrix agent.

The term "analytically inert matrix agent", when used in context of the present invention, relates to those molecular components in the host matrix that are inert, i.e. they do not have chemical activity, towards analyte molecules that are detected with the biosensing surface. These analytically inert matrix components can though have chemical activity towards each other or towards the analyte specific binding agents or towards the substrate.

The term "analyte specific binding agent", when used in context of the present invention, relates to those molecular components in the host matrix that have a specific activity towards the analyte molecules that are detected with the sensing surface. Analyte specific binding agent molecules usually comprise a body and a linker unit that binds specifically to a certain analyte molecule.

Now it has been surprisingly found out that by adding to the host matrix molecules binding units, which are able to form covalent bonds with the substrate surface, the mechanical, chemical and/or thermal stability of the monolayer, which form the sensing surface on the solid substrate, is enhanced. It is considered particularly surprising that even if only molecules of analytically inert matrix agent comprise these binding units, which covalently bind to the substrate surface, the lateral pressure of the monolayer is enough to keep also molecules of the analyte specific binding agent stabilised and oriented in the monolayer.

Typically a biosensing surface according to the present invention is formed by using Langmuir-Schaefer (LS) film-forming technique. The biosensing surface can be formed as one-step deposition, which is fast, simple and timesaving. According to one aspect of the present invention enhanced stability, reproducibility and hence reliability, as well as simultaneous fast production of the solid-supported monolayer can be achieved by the combined chemisorption and the LS film-forming technique. According to the present invention the Langmuir monolayer comprises agents, i.e. components, which react covalently with the substrate surface. The present invention thus results in a monolayer, which is not only physisorbed but chemically bound to the substrate surface. This increases mechanical stability of the film being built up of molecules with fixed position on the substrate. For the same reason, compared to conventional physisorbed films, the chemical stability of the film is increased, i.e. dissolution of the film-forming molecules by solvents is restricted. Thermal stability of the film is also increased, i.e. thermal motion of the substrate bound film-forming components is restricted.

According to one embodiment of the present invention the oriented monolayer, which is transferred to the surface of the solid substrate, binds covalently to the solid substrate also via binding units comprised in the molecules of the analyte specific binding agent of the host matrix.

According to one embodiment of the present invention suitable amphiphilic molecules, which can form the host matrix, are aliphatic fatty acids, alcohols or amides, or derivatives thereof. In other words these molecules can function as an analytically inert matrix agent or they can form the body of an analyte specific matrix agent into which the linker unit is attached. Chain length of a host matrix molecule is from 8 to 40 carbon atoms, typically over 10 atoms, more typically 14 to 20 atoms. Host matrix molecule chain can be straight or branched, saturated or unsaturated. Typical film forming host matrix molecule, i.e. analyte specific binding agent and/or analytically inert matrix agent, can be also phosphoglycerol or threitol derivatives which are esterified, etherified or amidated by aliphatic fatty acids, alcohols or amides, or one of their derivatives. For example phospholipids are good matrix molecules. According to one embodiment of the invention a binding unit is incorporated in to a number of host matrix molecules. Typically at least 50 %, more typically 80 - 95 %, of the host matrix molecules are incorporated with binding units that bind covalently to the solid substrate. Usually the binding units are comprised in the molecules of analytically inert matrix agent, but in certain cases also molecules of analyte specific binding agent can comprise binding units that bind covalently to the solid metal substrate.

The binding units that are used for forming the covalent bonds with the substrate surface can be chosen depending on the type of the substrate used. According to one embodiment of the invention the host matrix molecule structure with the binding unit is R-SH, R-S-R, or R-SS-R, the R symbolising an alkyl group of the host matrix molecule. For example, when gold, silver or copper is used as a substrate material, -SS- groups can be used as binding units. They react slightly slower with a gold surface than e.g. -SH groups, but they are chemically more stable, which makes the preparation of the sensing surface more reliable. According to one embodiment of the present invention the host matrix monolayer is thus bound covalently on to the solid surface via metal-specific binding units of analyte specific binding agent and/or analytically inert matrix agent.

The present invention not only improves the mechanical stability of the sensing surface, it also reduces the amount of analyte specific binding agent needed in the host matrix. As analyte specific binding agent is usually the most expensive component of the host matrix, the invention also reduces the preparation costs of the sensing surface. At the same time, as a small amount of the analyte specific binding agent is fairly evenly distributed in the host matrix, the binding of analyte molecules to the sensing surface is easier and more effective, as steric hindrance that they exert to each other is reduced. According to the invention only 2-15 mol-%, typically 5- 10 mol-% analyte specific binding agent is needed in the host matrix. According to one embodiment of the present invention the amount of analyte specific binding agent in the host matrix can be tailored according to e.g. size of the analyte molecule or unit to be detected. If the analyte molecule to be detected is large in size, the number of molecules of analyte specific binding agent is typically smaller in order to minimise the problems caused by steric hindrance. If the size of the analyte molecule is small, it is possible to have a larger number of molecules of analyte specific binding agent in the host matrix. In other words, the present invention enables the optimisation of amount of the analyte specific binding agent used.

According to one embodiment of the present invention the solid substrate, on to which the host matrix forming the biosensing surface is covalently bound, comprises a noble metal, such as gold, copper, platinum, palladium or silver. For example, when platinum is used as a substrate, alcohols and amines can be used as host matrix forming molecules. Noble metal used may be a single crystal, or it is evaporated to the support structure to form an ultra-flat surface, with a typical roughness of less than 1 nm/lμm². The support structure itself can be selected from a variety of materials, e.g. metal, glass, composite or synthetic polymer, mica or quartz. According to one typical embodiment the support structure is mica or glass.

According to one embodiment of the present invention the host matrix solution is polymerised by internal cross-linking at least of the molecules of the analytically inert matrix agent at the solid substrate or on the subphase surface, possibly before the transfer onto solid substrate. The stability of the sensing surface can be thus further enhanced by intermolecular cross-linking within a monolayer. This means that the host matrix molecules are polymerised to form a cross-linked network by internal cross-linking. For example, when diacetylene monomers are used in the host matrix they can be polymerised to polydiacetylenes. Also molecules of analyte specific binding agent can be cross-linked, either with each other, or with molecules of analytically inert matrix agent, or with both. According to one embodiment of the present invention the linker unit that is incorporated in the analyte specific binding agent molecules can be chosen depending on the analyte, which will be detected. For detection of biological analytes, the linker unit is chosen so that antibodies, hormones, allergenes, viruses, bacteria or other small organic molecules or units can be detected. According to one embodiment of the invention the linker unit in analyte specific binding agent in the host matrix is lipoate, biotin or maleimide. Suitable linker units for antibody binding are e.g. N-(ε-maleimidylcaproyloxy)succinimide (EMCS) or N- succinimidyl-3-(2-pyridyldithio)propionate (SPDP). A suitable linker unit for binding of streptavidin is e.g. biotin.

According to one embodiment of the invention the analytes that are bound to the sensing surface according to the present invention can be detected by quartz crystal microbalance as increase of the weight of the surface layer, or by surface plasmon resonance as increase in film thickness, or linker units may be incorporated with fluorescence labels for detection.

According to the invention the subphase used can be chosen depending on which kind of analytes are detected. For detecting bioanalytes, such as antibodies or antigens, biological buffers or water are preferred. The biosensing surface according to the invention can be used for detection of antibodies, hormones, allergenes, viruses, bacteria or other small organic molecules or units.

Depending on the components in the host matrix different solvents can be employed, such as chloroform, ethanol, cyclohexane or hexane.

A method for preparing a biosensing surface according to one embodiment of the present invention and the characterisation of the prepared biosensing surface is presented in the following non-limiting example. EXAMPLE

Materials and methods l-Palmitoyl-2-hydroxy-i'«-glycero-3-phosphocholine and l,2-dipalmitoyl-,s?2- glycero-3-phosphocholine (DPPC) were obtained from Avanti Polar-Lipids. Methyl methanethiosulfonate (Sigma), hydrobromic acid (49%, Sigma), 16- hydroxyhexadecanoic acid (Sigma), sodium hydride (60% dispersion in mineral oil, Aldrich), pyridine (Sigma), thioacetic acid (Lancaster), sodium hydroxide (J. T. Baker), ammonia solution (25%, Riedel-de Haen), hydrochloric acid fuming (37%, Merck), sodium chloride (J. T. Baker) and calcium chloride dehydrated (Fluka Chemika) were used as obtained. Dicyclohexylcarbodiimide (DCC, Acros Organics) and 4-dimethylaminopyridine (4-DMAP, Sigma) were recrystallised once from toluene (J. T. Baker) prior to use. Chloroform (Lab-Scan) and carbon tetrachloride (J. T. Baker) were dried over phosphorus pentoxide (Sigma). Ethanol (Primalco), hexane (Sigma), methanol (Merck), acetone (Shell) and diethyl ether (Fluka Chemika) all were of at least 99% purity. Human IgG and polyclonal goat anti-human F(ab')₂ were obtained from Jackson Immuno Research. F(ab )₂ was cleaved onto Fab fragments with dithiotreitol (DTT, Acros Organics) under argon overnight in a microdialysis tube prior to use [Ishikawa]. HEPES/EDTA buffer (10 uiM HEPES (Sigma), 150 mM NaCl (Fluka), 5 mM EDTA (Sigma), pH=6.8) was used in all antibody-antigen systems. Chromatographic separations were carried out by using precoated Merck 0.25-mm silica gel 60 TLC plates and Merck 70-230 ASTM silica gel. Detection on TLC plates was made by using iodine vapour. 1H NMR spectra were measured with Oxford Instruments 400 MHz Jeol JNM-GX- 4000 NMR-spectrometer. UV spectra were carried out using a Shimano UV 240/Visible spectrophotometer fitted with an OPI-4 computer interface. IR spectra were recorded on Perkin-Elmer Paragon 1000 FT-IR-spectrometer. Contact angles were measured by using water in ambient conditions. A Kriiss G-l goniometer attached to video camera, TV and VCR was used for all measurements. All angles were measured within 20 s after placing the water drops on each sample, at different locations on the surface of the gold slide.

Synthesis The linker lipid DPPGL was synthesised as previously described [Pax and Blume] and characterised with 1H NMR and UV/Vis-spectrometry. The structure of the end product is shown in Figure 1. The synthetic route of the host matrix lipid 1- palmitoyl-2-(16-(S-methyldithio)-hexadecanoyl)-j«-glycero-3-phosphocholine (DSDPPC) is outlined in Figure 1. Detailed description of the synthesis is given below (steps II- VI).

16-Bromohexadecanoic acid (II) [Bain et al] 16-hydroxyhexadecanoic acid (I) (2.38 g, 9.1 mmol) was refluxed for 49 h in a 1:1 mixture of 48% hydrobromic (15 ml) and glacial acetic acid (15 ml) in the dark. Upon cooling, the 16- bromohexadecanoic acid (II) crystallised as a white solid. The mixture was filtered through a Bϋchner funnel, washed several times with water, and recrystallised from ice-cold hexane. The yield was 2.65 g (87 %).

16-Mercaptohexadecanoic acid (III) [Bain et al] Sodium hydride (60% w/w in mineral oil) (0.7 g) was separated from mineral oil in the following way: NaH in mineral oil was dissolved in diethyl ether (30 ml). The mixture was strrred and the large portion of ether, which contains the mineral oil, was decanted away.

Remaining ether was quickly removed by rotary evaporation. Bromide (II) was converted to thioacetate by adding 50 ml of ice-cold methanol, 16- bromohexadecanoic acid (II, 2.6 g, 7.8 mmol) and thioacetic acid (1.18 ml, 16.6 mmol) into the flask, which contained previously separated sodium hydride and refluxing 19 hours. The addition of the thioacetic acid formed a turbid yellow mixture, which turned to a clear yellow solution after heating. After 19 hours, the solution was turned colourless and white solid was formed. After cooling to room temperature the thioester was hydrolysed by adding 60 ml of 1 M NaOH solution (degassed with N₂) and refluxing for 3 hours under nitrogen. The reaction mixture was then cooled with an ice-bath and poured with stirring into a beaker containing ice-cold water (200 ml), concentrated HC1 (10 ml) and diethyl ether (225 ml). The layer containing ether was separated, then washed with water (3 x 100 ml) and saturated aqueous NaCl solution (100 ml, 0.38 g / 1 ml), and dried over dehydrated CaCl₂. Finally, the mixture was filtered and the solvent was removed by rotary evaporation. The resulting white solid substance was recrystallised from ice-cold hexane and yielded 1.4 g (63 %) of 16-mercaptohexadecanoic acid. The product was storage at 0°C in the dark. TLC (2% MeOH in CHC1₃): R (product) = 0.40. 1H NMR (CDC1₃) δ 1.26 (s, 22 H, CH₂), 1.32 (t, 1Η, SH), 1.57-1.67 (m, 4Η, HSCH₂CH₂(CH₂)nCH₂), 2.35 (t, 2Η, HOOCCH₂CH₂), 2.52 (t, 2H, HSCH₂CH₂)

16-(S-methyldithio)-hexadecanoic acid (IV) [Samuel et al., Runquist and Helmpamp] A mixture of 16-mercaptohexadecanoic acid (1.22 g, 4.2 mmol), methyl methanethiosulfonate (0.65 ml, 6.3 mmol), and pyridine (0.51 ml, 6.3 mmol) in chloroform (13 ml) was stiπed at room temperature in the dark for 23 hours. 16- mercaptohexadecanoic acid dissolved poorly in chloroform, but the solubility increased as the 16-(S-methyldithio)-hexadecanoic acid was formed, and the solution was completely clear at the end of the reaction. Upon removal of the solvent by evaporation under reduced pressure, the residue was washed twice with ice-cold ethanol (the white-brown solid turned white) followed by recrystallisation in ice-cold hexane. The product was dried over P₂0₅ to yield 0.92 g (72 %) of 16- (S-methyldithio)-hexadecanoic acid. The product was stored at 0°C in the dark. TLC (2 % MeOH in CHC1₃): R_f (product) = 0.45. 1H NMR (CDC1₃) δ 1.26 (s, 22 H, CH₂), 1.57-1.72 (m, 4Η, SSCHzC^CCH^nCHz), 2.35 (t, 2H, HOOCCH₂CH₂), 2.41 (s, 3H, CH₃), 2.71 (t, 2Η, SSCH₂CH₂)

16-(S-methyldithio)-hexadecanoic acid anhydride (V). [Selinger and Lapidot] A solution of DCC (0.32 g, 1.52 mmol) in dry CC1₄ (5 ml) was added to a solution of 16-(S-methyldithio)-hexadecanoic acid (0.92 g, 3.04 mmol) in dry CC1₄ (30 ml). The reaction mixture was kept at room temperature under N₂ atmosphere and protected from light. After 18 hours the mixture was filtered by suction in order to remove N,N-dicyclohexylurea, which had formed. Examination of the filtrate by IR revealed the presence of 16-(S-methyldithio)-hexadecanoic acid anhydride (v_c=0 1752 and 1819 cm^"1) and absence of the parent carboxylic group (v_c=0 1702 cm^"1). The solvent was removed by evaporation under reduced pressure. The yield was 0.8 g (87 %). The product was dried over P₂0₅ and stored at -5°C in the dark.

l-Palmitoyl-2-(16-(S-methyldithio)-hexadecanoyl)-sn-glycero-3-phosphocholine (VI) [Runquist and Helmpamp, Mason et al, Mangroo and Gerber] l-Palmitoyl-2- hydroxy-£«-glycero-3-phosphocholine (0.33 g, 0.66 mmol) was suspended in 35 ml of dry chloroform and 4-DMAP (0.3 g, 2.45 mmol) and previously prepared anhydride (0.8 g, 1.32 mmol) was added to the mixture. The reaction mixture was stirred at room temperature under nitrogen atmosphere and protected from light. 1- Palrritoyl-2-hydroxy-s«-glycero-3-phosphocholine dissolved poorly in chloroform, but solubility increased as the l-palmitoyl-2-(16-(S-methyldithio)-hexadecanoyl)- st2-glycero-3-phosphochorine was formed, and the solution was completely clear at the end of the reaction. After 43 hours, the mixture was added to a separator funnel and diluted to 50 ml with chloroform. Then 35 ml of methanol and 20 ml of 0.1 M HC1 were added, and the lower phase was isolated. The upper phase was re- extracted with 2 x 20 ml of chloroform and the extracts were combined and the solvent was removed by rotary evaporation. The residue was precipitated by the addition of acetone-chloroform (95:5) and purified by column chromatography. The column was eluted with chloroform, chloroform-methanol (9:1), and chloroform- methanol-NH₃ (25%) (1:1:0.1). The product was precipitated by the addition of acetone-chloroform (95:5) and filtered by suction to yield 300 mg (60 %) of pure 1- palmitoyl-2-( 16-(S-methyldithio)-hexadecanoyl)-5^,w-glycero-3-phosphocholine. The product was identified with 1H NMR spectrum and UV spectrum. TLC (CHC1₃- MeOH-NH₃ (25%) (65:25:4): R_f (product) = 0.35. UV (CHC1₃): λi = 250 nm. 1H NMR (CDC1₃) δ 0.88 (t, 3H, CH₃(CH₂)ι₂, 1-25 (s, 46H, CH₂), 1.54-1.60 (m, 4Η, OOCCH₂CH₂), 1.65-1.72 (m, 2Η, SSCH₂CH₂), 2.26-2.33 (m, 4H, OOCCH₂), 2.41 (s, 3H, CH₃SS), 2.70 (t, 2H, SSCH₂), 3.39 (s, 9Η, N⁺(CH₃)₃), 3.88 (m, 2Η, POCH₂CH₂), 3.93-4.03 (m, 2Η, CHCH₂OP), 4.10-4.15 (m, 1Η, CH₂CΗCΗ₂OP), 4.36-4.40 (m, 3H, POCH₂CH₂ and CH₂CHCH₂OP), 5.18-5.23 (m, 1H, CHCT OP).

Surface pressure measurements

A commercially available computer-controlled KSV LB-5000 Langmuir trough (KSV-Instruments, Helsinki, Finland) with a Wilhelmy balance was used. The Milli Q filtration system (Millipore Corp.) was used to purify the water for the subphase having a resistance of 18 MΩcm. DSDPPC was mixed in various ratios with the linker lipid (DPPGL) in chloroform to produce a solution with concentration of 1 mg/ml and spread onto the surface of subphase using microsyringe. The solvent evaporated during 10-20 min leaving a monomolecular layer of lipid on the subphase surface. Films of this material were compressed at 5 mm/min, to produce surface pressure vs. mean molecular area isotherms. The experiments were carried out at temperatures 10 - 31 °C.

Surface potential measurements

The surface potential measurements for the pure lipids (DSDPPC, DPPGL and DPPC) were performed with a LB-5000 Langmuir trough. The experiments were carried out at 20 ± 0.4°C. The surface potential, ΔV, of the monolayer was measured simultaneously with the surface pressure by the vibrating plate method.

The upper, vibrating Pt electrode was positioned about 2 mm above the subphase surface, and it was perforated to minimise the noise.

LS deposition of the lipid monolayers onto a gold substrate

Ultra-flat gold surfaces for use as substrates for horizontal deposition were prepared following the procedure described by Wagner et al. The mixed film of DSDPPC and DPPGL was prepared as described in surface pressure measurements and the monolayer was compressed to a predetermined pressure. The gold substrate was then brought into contact with the floating monolayer for a certain period of time. After lifting up the lipid-coated substrate was washed with high-purity water and absolute ethanol and dried with nitrogen. A schematic illustration of the horizontal deposition procedure is shown in Figure 2.

Scanning probe microscopy (SPM)

The samples for SPM imaging were prepared in the following way. The gold substrates with the LS-deposited binary lipid monolayers were kept in a Hepes- EDTA buffer solution of Fab fragments for 2 h, followed by 18 h in a BSA solution at 4 °C and subsequently 2 h in a hlgG solution. Substrates were gathered from each coating step for SPM imaging, rinsed with buffer and high-purity water and dried with nitrogen. The slides were kept dry at room temperature until the imaging was performed.

A Nanoscope Ilia (Digital Instruments, Inc., Santa Barbara, CA) SPM in tapping mode was used for imaging the sample surfaces in ambient air. A J-scanner (150 x 150 μm scan range) and silicon cantilevers (TESP) supplied by the manufacturer (Nanoprobes TM) were used for imaging. The free amplitude of the oscillating cantilever (off contact) was 60 nm. The engage procedure which caused a shift in the resonance frequency was taken into account and the new resonance frequency for the tip in contact was determined and used as the operating frequency. Light tapping, with a damping ratio (contact amplitude/free amplitude) of about 0.7-0.8 was used for imaging. All images were collected in the height and phase mode. The SPM imaging was accomplished within 30 min from the sample preparation.

Pressure-area and Surface Potential Isotherms of Pure DSDPPC and DPPGL The surface pressure-area isotherms of pure DSDPPC on pure water subphase at different temperatures shown in Figure 3 clearly show that DSDPPC forms a monolayer with a characteristic temperature-dependent liquid-expanded to liquid- condensed phase transition (LE-LC). This LE-LC phase transition is apparent between temperatures 12.2 °C and 23.7 °C. For comparison, the phase transition temperature for DPPC is 41.5 °C [Pax and Blume]. As earlier reported by Ihalainen and Peltonen, DPPGL is also surface active and has a LE-LC phase transition below the crystal melt point T_m (22.9 °C). Above T_m the monolayer remains in the LE state throughout the compression [Ihalainen and Peltonen]. Figure 4A shows that at 20.0 ± 0.4 °C DSDPPC collapsed at a surface pressure of 43 mN/m. This value is lower compared to DPPC, shown in Fig. 4C, and DPPGL, shown in Fig. 4B, which both collapsed at a surface pressure of 55 mN/m. The results show that the terminal disulphide group introduced in the alkyl chain decreases the collapse pressure. However, the comparison of extrapolated mean molecular area (A_ext) values of compressed monomer films of DSDPPC (0.45 run²) and DPPC (0.49 nm²) in Table 1 shows that the disulphide group enhances the condensation of the monolayer. This indicates that the space filling of DSDPPC is more effective than that of DPPC, most probably because of the unequal length of the alkyl chains of DSDPPC. This presumably leads in a more perpendicular orientation of the lipids as compared with DPPC. This means that DSDPPC reaches a preferable orientation considering the gold-sulphur adsorption process.

Table 1. Surface potentials and effective dipole moments of phospholipid mono- layers and their head group regions at the water/air interface at 20 + 0.4°C.

MONO- A_ext(nm²) ΔV_max (mV) ΔV_α (mV) μ_n (D) μ_α (D) LAYER

DSDPPC 0.45 +402 +109 +0.48 +0.13

DPPC 0.49 +625 +85 +0.81 +0.11

(+669)^a (+99)^a (+0.82)^a (+0.12)^a

DPPGL 0.42 +462 -161 +0.52 -0.18

^aValues taken from Vogel and Mobius The surface potential isotherms followed nicely the changes in the compression isotherms and were reproducible throughout the entire area range studied. Typical ΔV-A isotherms for DSDPPC, DPPC and DPPGL are also shown in Figure 3. The obtained ΔV_max values for DSDPPC, DPPC and DPPGL were 402 mV, 625 mV and 462 mV.

The surface potential for a monolayer at the air-water interface can in its very simplest scheme be expressed by the Helmholtz equation as

ΔV = μ εεoA where μ_l is the normal component of the molecular dipole moment, ε is the relative permittivity of the monolayer, and ε₀ is the permittivity of vacuum. According to Vogel and Mobius the total effective dipole moment _x can be divided into two parts, μ_a_ ά μ^ω. All contributions associated with the polar headgroup including the reorganisation of water molecules near the lipid monolayer interface are represented by μ_a, whereas μ^ω represents the effective dipole moment of the hydrophobic part of the monolayer. If we assume that ε= l and use the effective dipole moment value +0.35 D for a CH₃-group of a hydrocarbon chain aligned parallel to the normal of the surface within a close-packed monolayer [Vogel and Mobius], we can estimate from surface potential data the effective dipole moment of the hydrated polar head groups of the phospholipids studied here. The contributions of the monolayer/air and the monolayer/water interfaces to the total surface potential are compiled in Table 1. A_ext represents the extrapolated mean molecular area, where the monolayer appears in the most condensed state. The CH₂-groups of the hydrocarbon chains do not contribute to the total dipole moment [Habib and Bockris]. The zwitterionic phosphocholines have relatively small effective dipole moments of +0.13 D for DSDPPC and +0.11 D for DPPC, whereas for DPPGL the value is -0.18 D. The effective dipole moment of the SSCH -group was calculated within the limits of error to be close to zero. Pressure-area Isotherms of Binary Monolayers

Surface pressure-area isotherms of binary mixtures of DSDPPC and DPPGL measured at 20.0 ± 0.4°C are shown in Figure 5. The clear LE-LC phase transition present in the isotherms of the pure components disappears when the linker molecule is introduced in the mixture, but reappears at higher linker concentrations and is clearly present in the mixture ratio of 50:50 mol-%. At low DPPGL concentrations, the mixed monolayers collapsed at the same surface pressure as pure DSDPPC, 43 mN/m, but at higher linker mole ratios, over 5 mol-%, the mixed monolayers collapsed at the same surface pressure as pure DPPGL, 55 mN/m. Also the extrapolated mean molecular areas of the compressed monomer films followed the same trend. At low linker mole ratios, the A_ext is the same as that of pure DSDPPC, 0.45 nm², and again when the linker concentration is increased over 5 mol-%, the A_ext is the same as pure DPPGL, 0.42 nm . This indicates that the linker is miscible with DSDPPC at a low mole ratio only, and at higher mole ratios the two molecules are phase separated. This phase separation could also rise from the fact that pure DSDPPC and DPPGL have very different phase transition surface pressures, as seen in Figure 5.

The excess area criterion [Gaines, Birdi] to the binary monolayers was also tested to study the miscibility of the components in the monolayers. Figure 6 shows the average molecular area at surface pressures of 5, 15, 25 and 35 mN/m of the mixed

DSDPPC/DPPGL monolayer as a function of the molar fraction of DSDPPC. The dotted lines in Figure 6 demonstrate the deviation of the data points from the linear dependence of molecular area on the mixing ratio of the film components. The lines are drawn between data points of pure DSDPPC and DPPGL and represent the ideal mixing behaviour. The large deviations from the linear dependence at the higher surface pressures when over 20 mol-% of linker component is present in the mixture indicates that DSDPPC/DPPGL monolayer seems to be miscible when the linker lipid is in clear minority (< 15 mol-%). Scanning Probe Microscopy (SPM) measurements of pure DSDPPC monolayers on a gold-coated substrate.

Figure 7 shows typical SPM height images of the pure gold substrate (Fig. 7A), and DSDPPC monolayer deposited onto the gold-coated substrate using LS-deposition at the surface pressure of 30 mN/m at 20.0 ± 0.4 °C, Fig. 7B - 7F. Gold substrates were kept in contact with the Langmuir monolayer for 20 s (Fig. 7B), 1 min (Fig. 7C), 3 min (Fig. 7D), 15 min (Fig. 7E) and 2 h (Fig. 7F). Images show that the gold substrate is almost fully covered with lipid molecules already after 20 s of adsorption. The dark holes in the height image represent the gold surface with no DSDPPC molecules adsorbed. In a 20-s, 1-min and 3-min samples there is more holes in the monolayer and they are larger as compared with the 15-min and 2-hour samples. The cross section analysis gave an intermediate depth of the holes of 1.6 - 2.9 nm for every other but the 2-hour sample. In the 2-hour sample there were practically no visible holes, the substrate was fully covered with a lipid monolayer. The intermediate monolayer thickness values of 1.6 - 2.9 nm corresponds to the reported experimental thickness value of 1.77 nm of a self-assembled monolayer of 1 -palmitoyl2-( 16-mercaptohexadecanoyl)5^,n-glycero-3phosphocholine spontaneously adsorbed on gold, and to the predicted maximal length of 2.8 nm of the same phosphocholine, calculated from space-filling models (CPK) [Diem et al.]. The root-mean-square roughness (R_τms) value over a lxl μm² area of the lipid monolayer on a gold substrate varied according to the adsorption time and are presented graphically in Figure 8. These results show that the monolayer adsorption from the surface is very fast as expected, the monolayer reaches its final homogeneity after 2 hours. On the basis of the results it was decided to use an adsorption time of 2 hours for further protein adsorption studies.

The measured contact angle values against water of the gold substrate coated with a

DSDPPC monolayer also varied slightly according to the adsorption time as shown in Figure 8. Although the DSDPPC coated gold substrate was somewhat more hydrophilic than the pure gold surface, the contact angle values show that DSDPPC molecules were adsorbed in a somewhat tilted orientation with respect to the surface normal, thus decreasing the hydrophilicity of the surface, especially regarding the short adsorption times. The contact angle value of the gold substrate coated with DSDPPC monolayer is close to the value 47 ± 6° measured for l-palmitoyl-2-(16- mercaptohexadecanoyl)-s«-glycero-3-phosphochorine on gold [Diem et al].

SPM measurements of protein immobilisation to the binary films Figure 9 shows typical SPM height images of the binary monolayers of DSDPPC/DPPGL in two different compositions, 95:5 mol-% (Fig. 9A) and 80:20 mol-% (Fig. 9B), deposited at surface pressure 30 mN/m and kept on the subphase surface for 2 hours. The images show that in both cases the gold substrate is fully covered with a monolayer and there are no large holes visible, neither any signs of the phase separation.

The contact angle value for the 95:5 mol-% mixture film was 55 + 5° and for the 80:20 mol-% monolayer 63 ± 5°, respectively. The slightly higher values than those obtained for pure DSDPPC monolayer, indicate that the binary monolayers are less organised and homogeneous than the pure DSDPPC monolayers. However, the smaller contact angle value of the 95:5 mol-% mixture as compared with the 80:20 monolayer indicates that increasing the concentration of the linker in the binary system results in a more inhomogeneous surface. Also the R_rms- values over a 2 x 2 μm area, 0.31 nm for the 95:5 and 0.41 nm for the 80:20 mixed film, support this conclusion.

SPM was further used to image the binding of Fab' fragments and antibody-antigen complex formation onto DSDPPC/DPPGL matrices (Figure 10) with the fractions of the lipids being 95:5 in Fig. 10A, Fig. 10C and Fig. 10E, and 80:20 mol-% in Fig. 10B, Fig. 10D and Fig. 10F. Fab' fragments were immobilised from a Hepes- EDTA buffer solution with a concentration of 30 μg/ml and the R_rms values were determined in order to obtain comparable information about the immobilisation process, shown in Table 2.

Table 2. R_rms values over area of 2 x 2 μm² after the different immobilisation steps measured by SPM.

R_rms (run)

MONOLAYER 95:5 mol-% 80:20 mol-%

PHOSPHOLIPID 0.31 0.41 MONOLAYER

Fab' 1.21 1.86

BSA 0.52 0.67

hlgG 1.60 1.10

There was a clear change in the R_rms value as a result of Fab fragment binding, i.e. in the 95:5 mol-% mixture the R_rms value increased by 0.90 nm to 1.21 nm and for the 80:20 mol-% film the increase was 1.45 nm to 1.86 nm. The higher initial R_rms value of the 80:20 mol-% mixture, however, contributed to the fact that the Fab' fragments formed more and larger aggregates on the matrix system. This was apparent from the SPM height images (Fig. 10A and 10B), where globular objects were observed. For the 95:5 mol-% mixture the diameter of the globular object varied between 40-86 nm, whereas for the 80:20 mol-% film the corresponding value was 80-200 nm. The dimensions of the globular objects for the 95:5 mol-% film correspond to the value 20-80 nm reported recently by Vikholm et al. Also the measured height of the Fab' fragments, the values being 4.0-6.0 nm for the 95:5 mol-% mixture and 5.1-7.4 nm for the 80:20 mol-% mixture coπesponded well to the Fab' fragment dimensions 7x5x4 nm³ measured by x-ray diffraction [Sarma et al.]. On the basis of these finding and if we take into account the fact that the tip- sample convolution and distortion of the molecules under scanning tip usually tends to affect, i.e.increase, the imaged size of the objects [Elender et al], the majority of the objects in the case of 95:5 mol-% mixture are believed to be Fab '-fragments bound to the DSDPPC/DPPGL matrix in a side-on (4 nm), edge-on (5 nm) or standing (7 nm) but slightly tilted orientation.

The exposure of the surface to 0.1 mg/ml BSA in Hepes-EDTA buffer solution decreased the R_rms value for both compositions (Table 2). This was expected, because BSA has no specific affinity to lipid layers, although it does adsorb to almost any surface [Golander and Kiss]. The decrease in the R_rms values indicates that the BSA molecules had adsorbed onto the surface where the lipid matrix was free from Fab' fragments. That is why the height images, Figures IOC and 10D, look much smoother than the surfaces with only Fab' bound, Figure 10A and 10B. Consequently, most probably BSA did not adsorb on the Fab' fragments.

When the hlgG molecules were introduced to surface, Figures 10E and 10F, the R_rms values increased considerably for both film structures as seen in Table 2. The increase in the R_rms value was however smaller in the 80:20 system than in the 95:5 system, indicating that the surface concentration of the immobilised antibodies was higher in the former case, as stated earlier. This decreases the interaction with hlgG due to steric hindrance. The average height of the adsorbed hlgG molecules was 7.0-9.0 nm for the 95:5 and 5.0-8.0 nm for the 80:20 system. Although the dimensions of both the Fab' fragments and the hlgG molecules depend on the orientation of the Fab' fragments in the hinge region, it is seen from the height values that hlgG molecules are adsorbed in a somewhat tilted orientation on the surface.

The present invention thus increases the mechanical, chemical and thermal stability of the sensing surface and makes the preparation of such sensing surfaces by using the Langmuir-Schaefer technique easy and simple. The present invention can therefore be used to increase the possible uses of highly organised LS monolayers as sensing surfaces in different sensor applications. The present invention allows, for example, preparation of a sensing surface, which offers a promising way to achieve a site-directed immobilisation of antibodies with high antigen-binding efficiency. The terminal binding unit introduced in the host matrix molecules decreases the collapse pressure and enhances the condensation of the monolayer. The invention can be used also in other sensing surface applications than in biosensing, for example in sensing of inorganic molecules and compounds.

The present invention provides, among other things, a possible way to include a functional unit, i.e. analyte specific binding agent, into a film that forms a sensing surface, without reducing mechanical stability. The mechanical stability of the film is not essentially reduced, because the lateral pressure of the film is enough to keep the molecules of the analyte specific binding agent in the film, even if those molecules themselves would not be directly bound to the substrate surface, and because the amount of analyte specific binding agent needed can be kept on a low level.

The present invention also provides a way to include a functional unit, i.e. analyte specific binding agent, into a film that forms a sensing surface, without phase separation of the film components. According to one embodiment of the invention the phase separation does not occur even if a different lipid is comprised in the film forming host matrix, because the apolar parts of the analytically inert matrix agent and the analyte specific matrix agent are structurally similar.

It will be appreciated that the essence of the present invention can be corporated in the form of a variety of embodiments, only a few of which are disclosed herein. It will be apparent for the specialist in the field that other embodiments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as restrictive. REFERENCES

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Claims

1. Method of preparation of a biosensing surface comprising the following steps:

- compressing the monolayer, - bringing a solid metal substrate in contact with the monolayer on the subphase surface, and

- transfeπing the monolayer onto the solid substrate by using hydrophobic interactions between the monolayer and the solid substrate,

2. Method according to claim 1, characterised in that the monolayer binds covalently to the solid substrate via binding units comprised in the molecules of the analyte specific binding agent of the host matrix.

3. Method according to claim 1, characterised in that the host matrix is polymerised by internal cross-linking at least of the molecules of the analytically inert matrix agent on the subphase surface or at the solid substrate.

4. Method according to claim 1, characterised in that typically at least 50 %, more typically 80 - 95 %, of the host matrix molecules are incorporated with binding units that bind covalently to the solid substrate.

5. Biosensing surface, comprising - a solid metal substrate coated on a support structure, - an oriented host matrix monolayer on the solid substrate, the said monolayer

- bound covalently to the solid substrate via binding units comprised in the i molecules of the analytically inert matrix agent of the host matrix.

6. A biosensing surface according to claim 5, characterised in that the monolayer is bound covalently to the solid substrate via binding units comprised in the molecules of the analyte specific binding agent of the host matrix.

7. A biosensing surface according to claim 5, characterised in that the solid substrate is a noble metal, such as gold, silver, copper, platinum or palladium.

8. A biosensing surface according to claim 5, characterised in that the binding unit is R-SH, R-S-R, or -SS-, more typically R-SS-R, the R symbolising an alkyl group of the host matrix molecule.

9. A biosensing surface according to claim 5, characterised in that the analytically inert matrix agent in the host matrix is aliphatic fatty acid, alcohol or amide, or a derivative thereof.

10. A biosensing surface according to claim 5, characterised in that the linker unit in analyte specific binding agent in the host matrix is lipoate, biotin or maleimide.

11. A biosensing surface according to claim 5, characterised in that the analyte specific binding agent and/or analytically inert matrix agent in the host matrix is phosphoglycerol or threitol derivative, which is esterified, etherified or amidated by aliphatic fatty acid, alcohol or amide, or one of their derivatives.

12. A biosensing surface according to claim 5, characterised in that the length of the host matrix molecule chain is from 8 to 40 carbon atoms, typically over 10 atoms, more typically 14 to 20 atoms.

13. A biosensing surface according to claim 5, characterised in that the host matrix molecule chain is straight or branched.

14. A biosensing surface according to claim 5, characterised in that the host matrix molecule chain is saturated or unsaturated.

15. Use of a biosensing surface according to any of the claims 5 to 14 for detection of antibodies, hormones, allergenes, viruses, bacteria or other small organic molecules or units.