Polymer for binding amine containing ligands and uses thereof

Details Polymer for binding amine containing ligands and uses thereof Polymer for binding amine containing ligands and uses thereof

2002-04-22

2004-01-20

Snay, Jeffrey (Department: 1743)

Chemistry: analytical and immunological testing

Nitrogen containing

Amine and quaternary ammonium

C436S161000, C436S172000

Reexamination Certificate

active

06680204

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a polymer capable of binding amine ligands, to its preparation, and to uses including assays and chromatography.
There is an ever-increasing need for the measurement and quantification of mankind's activities. Sensors are partially fulfilling this requirement though sensors for many applications are not readily available in a form suitable for commercialisation.
One particular class of sensors, that is bio- and chemo-recognition sensors, requires the integration of ligands (recognition elements) to a transducer. The integration should normally enable changes in the recognition element caused by interaction with a corresponding analyte to be converted into a measurable signal via the transducer.
The immobilisation of the ligands to the surface of a suitable transducer is a necessary step in sensor manufacturing. Desirable properties of this process include: placement of appropriate amounts of ligands on detector surface using simple procedures, maintenance of biorecognition activity of the ligands after immobilisation, and minimisation of non-specific interactions between the sample and the recognition element or other parts of the sensor.
BACKGROUND ART
Recently a wide variety of methods have been discovered for attaching different ligands to transducers in order to create effective and sensitive bio- and chemosensors.
Examples are disclosed in U.S. Pat. No. 4177038, U.S. Pat. No. 4250267, U.S. Pat. No. 4784962, U.S. Pat. No. 5242828, U.S. Pat. No. 5436161 and Löfås, S., Johnsson, B., Tegendal, K., Rönnberg, I., “Dextran modified gold surfaces for surface plasmon resonance sensors: immunoreactivity of immobilised antibodies and antibody-surface interaction studies”, (1993), Colloids and Surfaces B: Biointerfaces, 1: 83-89.
Also of interest is Simons, S., Jr. et al. “Reaction of O-Phthalaldehyde and Thiols With Primary Amines; Formation of 1-Alkyl (and Aryl) Thio-.alpha.-Akylisoindoles”, (1978), J. Org. Chem., 43 (14): 2886-2896.
A common approach is the use of transducer surfaces modified with hydrogel layers (U.S. Pat. No. 5436161), such as carboxymethyl-dextran. Typically, hydrogel polymers are covalently immobilized to a transducer surface to form a thin hydrogel layer. Ligands can be covalently immobilised to the hydrogel polymer using a chemical activation. Such multi-step process places a significant overhead cost on any mass-manufacture of a sensor device. An alternative approach, which can eliminate this disadvantage, would be of considerable benefit.
DISCLOSURE OF INVENTION
In a first aspect the invention provides a method of immobilising an amino-group containing ligand L—NH
2
by means of a dialdehyde component OHC—X—CHO and a polymerisable component R—Z wherein R is a polymerisable moiety and Z is selected from —SH, —S-alkyl, —CN and —SO
2
by carrying out the following reactions simultaneously and/or sequentially in any chemically feasible order:
i) polymerisation of the polymerisable moieties R, optionally together with one or more comonomers;
ii) reaction of a component containing —Z with the dialdehyde component;
iii) reaction of L—NH
2
with the dialdehyde component or with the product of reaction (ii).
The dialdehyde component is preferably a 1,4-dialdehyde, generally conjugated
generally forming part of an aromatic ring system, e.g. it may be o-phthaldialdehyde (I):
Conjugation may be desirable to give a product detectable by optical methods, e.g. involving fluorescence.
The polymerisable moiety R generally contains one or more carbon-carbon multiple bonds. For example, R—Z may be allyl mercaptan.
Polymerisation may involve comonomers as well as the R moieties. They can be used to ‘dilute’ the Z groups in the polymer. Particularly when Z is —SH, such dilution may be desirable to reduce the occurrence of cross-linking through Z—Z (e.g. disulphide) bridges. Comonomers can also be used to regulate polymer solubility, to suit particular applications.
Particularly when Z is —SH or —S-alkyl, the reaction may be suitable for achieving self-assembly of a polymer on a suitable surface (particularly a metal surface (especially noble metal) or a surface having —SH groups). Thus a preferred class of embodiment is based on the ability of a synthetic polymer, containing thioacetal groups formed by a mercapto group and phthalic dialdehyde to self-assemble on a metal surface and bind amino containing ligands to form a fluorescent complex. The polymer can be synthesised using ion, radical polymerisation or polycondensation. It is preferable that at least one of the monomers used for polymerisation contains free SH groups. In another variant polymer can be first produced using an SH-group containing monomer and subsequently treated with dialdehyde to form a thioacetal. IL is also possible to use CN and SO
2
-containing monomers instead or simultaneously with SH-containing monomer for the polymer preparation.
Surfaces coated with a polymer of the invention may be microtitre plates, wells, transducers (e.g. for surface plasmon resonance or electrochemical devices), or binding materials e.g. for chromatography.
A polymer of the invention may comprise units of formula II:
where R′ is derived from polymerisation of an R group. The unit
is preferably provided by an aromatic ring system, e.g. being
optionally substituted and/or fused to form a polycycle. A polymer of the invention incorporating L—NH
2
may comprise units of formula III:
where
corresponds to
in formula II, e.g. being
(optionally substituted and/or fused.
The polymers described above can be used in affinity chromatography or sensors. Such a polymer containing SH groups may adsorb on a metal or SH-containing surface (e.g. during the synthesis) forming a homogeneous, stable coating. It is possible to make first a surface coating with a polymer of RSH, with following treatment of this polymer using dialdehyde, preferably phthalic dialdehyde and next amino-containing ligand. Again, ligand can be immobilised by simultaneous addition of the desired compound with dialdehyde to the SH-containing polymer. Species, such as cells, enzymes, viruses, fungi, antibodies, proteins, peptides, amino acids, nucleic acids and derivatives or mixtures can be easily immobilised on the polymer, synthesised as described above. The immobilised species may itself serve for binding a second type of ligand. Binding the second type of ligand may affect measurable properties, e.g. fluorescence, so that the binding may be detected. The ligands of first and second type may constitute specific binding pairs, e.g. antibody-antigen.
Ligand immobilisation on the polymer surface includes one-step interaction between thioacetal and amino group without chemical activation of the polymer or ligand functional groups. Formation of fluorescent complexes can be used to monitor binding. The isoindole complex formed by thioacetal with ligand amino groups was found to be very stable, which permits the use of strong acidic conditions for surface regeneration.
The homologous aromatic dialdehyde, o-phthaldialdehyde, or thioacetal is essentially nonfluorescent until reacted with primary amine in the presence of excess cyanide or mercaptan to yield a fluorescent isoindole. Monitoring of the polymer fluorescence provides an opportunity to directly control the amount of the bound substances to the polymer surface, which can be further used in sensors and assays.


REFERENCES:
patent: 4177038 (1979-12-01), Biebricher et al.
patent: 4250267 (1981-02-01), Hartdegen et al.
patent: 5436161 (1995-07-01), Bergstroem et al.
patent: 5981734 (1999-11-01), Mirzabekov et al.
patent: 6562581 (2003-05-01), Law et al.
patent: 0 233 973 (1987-09-01), None
patent: 4-323209 (1992-11-01), None
patent: WO 93/02115 (1993-02-01), None
I.H.S. Al-Deen et al, “Assay of picomole quantities of polyamines by high pressure ion-exchange chromatography” Proceedings of the B.P.S., Sep. 13th-15th 1978, p. 465P.*
B. Tamami et al, “Poly(vinyl pyridine) supported silver dichromates as versatile, mild and efficient oxidants for different o

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