WO2017029194A1 - Biocapteur à régénération - Google Patents

Biocapteur à régénération Download PDF

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Publication number
WO2017029194A1
WO2017029194A1 PCT/EP2016/069164 EP2016069164W WO2017029194A1 WO 2017029194 A1 WO2017029194 A1 WO 2017029194A1 EP 2016069164 W EP2016069164 W EP 2016069164W WO 2017029194 A1 WO2017029194 A1 WO 2017029194A1
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Prior art keywords
streptavidin
biotin
sensor chip
sensor
desthiobiotin
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PCT/EP2016/069164
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English (en)
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Hermann Gruber
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Johannes Kepler Universität Linz
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Publication of WO2017029194A1 publication Critical patent/WO2017029194A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/04—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D233/28—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D233/30—Oxygen or sulfur atoms
    • C07D233/32—One oxygen atom
    • C07D233/34—Ethylene-urea
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2610/00—Assays involving self-assembled monolayers [SAMs]

Definitions

  • the present invention relates to the field of sensors, particularly biosensor chips for detecting an analyte in a sample.
  • the sensor chip comprises a substrate layer and one or more organic conjugates bound to the substrate in form of a self- assembled monolayer (SAM).
  • SAM self-assembled monolayer
  • the present invention relates as well to a novel conjugate used to form a self-assembled monolayer (SAM).
  • SAM self-assembled monolayer
  • the present invention relates also to methods for regenerating the biosensor chip, thus enabling the repeated use the biosensor chips.
  • SAMs Self-assembled monolayers
  • SAMs are ordered molecular assemblies formed by the adsorption of amphiphilic, surfactant-type molecules on surfaces. SAMs provide one simple route to functionalize surfaces by organic molecules containing free anchor groups. The monolayer produced by self-assembly allows tremendous flexibility with respect to several applications depending upon their terminal functionality
  • biotin and avidin or streptavidin have been exploited for use in many protein and nucleic acid detection and purification methods.
  • Avidin-biotin binding is the strongest known non-covalent interaction between a protein and ligand.
  • the bond formation between biotin and avidin is very rapid, and once formed, is unaffected by extremes in pH, temperature, organic solvents and other denaturing agents.
  • These features of avidin make detecting or purifying biotin-labeled proteins or other molecules particularly useful for a number of biomedical applications.
  • a disadvantage of the strong binding is that it is essentially irreversible under physiological conditions.
  • Fig. 1 An acid-sensitive mutant of avidin serves as bridge between a biotinylated chip and biotinylated sensor molecules.
  • the avidin mutant M96H is rapidly dissociated into four denatured subunits by the combination of citric acid and SDS (Pollheimer et al., 2013).
  • the problem of nonspecific protein and DNA adsorption towards avidin M96H was solved by additional mutations which lowered the isoelectric pH (pi) to 7 (Taskinen et al., 2014) but these caused increased sensitivity to acid, so that the "standard regeneration" step (pH 2.5) for antibody-antigen interaction (Fig.
  • streptavidin as such from the desthiobiotin surface is a necessary but insufficient criterion for chip regeneration: Statistically biotinylated proteins (such as antibodies) can crosslink adjacent streptavidin molecules on the chip surface, preventing chip regeneration, even if the interaction between streptavidin and the chip is abolished (Pollheimer et al., 2013; compare also Fig. 8C and 8E).
  • biosensor comprising a self-assembled monolayer formed by a novel compound and a method for
  • biosensor chips biosensing methods and comparable heterogeneous assay formats in which biotinylated sensor molecules (e.g., antibodies) are stably immobilized on (strept)avidin-functionalized surfaces and cognate analyte molecules (e.g., antigens) are reversibly bound to the sensor molecules.
  • biotinylated sensor molecules e.g., antibodies
  • analyte molecules e.g., antigens
  • the purpose is either detection and quantification of the analyte molecules in a liquid sample, or biological interaction analysis between sensor molecules and analyte molecules (Fig. 1AB).
  • A is a moiety which provides for stable anchoring to a solid surface
  • n 0 or an integer of 1 to 22
  • X is selected from the group consisting of ether, thioether, ester, amide, urethane, urea, hydrazone, oxime, acetal bond, and a triazole product formed from azide with alkyne,
  • L denotes a hydrophilic polymer
  • Y is selected from the group consisting of ether, thioether, ester, amide, urethane, urea, hydrazone, oxime, acetal bond, and a triazole product formed from azide with alkyne, and
  • D denotes desthiobiotin or a derivative
  • the total linear chain of— (CH2)n— X— L— Y— comprises at least 27 backbone atoms.
  • a sensor chip comprising a substrate and a self-assembled monolayer comprising a compound of Formula I.
  • a method for detecting an analyte molecule in a sample comprising the steps of:
  • Fig. 1 is a schematic representation of the regeneration of a sensor chip.
  • Fig. 2A depicts various SAMs.
  • "db” stands for desthiobiotin.
  • Fig. 2B depicts examples of suitable hydrophilic polymers.
  • Fig. 3 is a schematic representation of the binding of streptavidin to SAMs with different lengths of hydrophobic segments (tilted) and hydrophilic segments (vertical).
  • Fig. 4 is a graph reporting the denaturation temperature of horse heart metmyoglobin in EPPS buffer at various concentrations of biotin or urea.
  • Fig. 5 is a sensorgram measured by surface plasmon resonance (SPR), showing imperfect chip regeneration by pulses of 200 mM biotin (pH 8.0).
  • the mixed SAM contained 20% component 9 and 80% component 8, as illustrated in Fig. 1 B.
  • Fig. 6 is an SPR sensorgram showing perfect chip regeneration by sequential injection of guanidinium thiocyanate (GTC), pepsin, and SDS (two cycles).
  • GTC guanidinium thiocyanate
  • pepsin pepsin
  • SDS two cycles
  • Fig. 7 is a schematic representation (panel A-D) and the corresponding SPR sensorgram (panel E) showing easy removal of streptavidin plus mono-biotinylated bovine serum albumin (BSA) by GTC without the need for a protease.
  • the mixed SAM was the same as in Fig. 5.
  • FIG. 8 is a schematic representation (panel A-D) and the corresponding SPR sensorgram (panel E) showing the need for protease action if streptavidin is
  • the mixed SAM was the same as in Fig. 5.
  • Fig. 9 is a measured SPR sensorgram demonstrating much slower chip regeneration by all kinds of reagents (GTC, pepsin, biotin) if the mixed SAM (80% component 8) contains 20% component 10 instead of 20% component 9, i.e., if the tether between the SAM surface and desthiobiotin is much longer (Fig. 3D) than the minimal requirement (Fig. 3B).
  • Fig. 10 is a measured SPR sensorgram demonstrating very unstable binding of streptavidin if the fraction of component 9 is lowered to 1 % (as compared to 20% in Fig. 5-8), i.e., if most streptavidin molecules are bound to one desthiobiotin residue only (see Fig. 3C).
  • Fig. 1 1 is a measured SPR sensorgram proving absence of protein adsorption both before and after coating of the mixed SAM with streptavidin.
  • the mixed SAM was the same as in Fig. 5.
  • Fig. 14 is a graph showing the data evaluation from Fig. 13 by the "double referencing method” (solid lines, obtained by subtraction of FC2 from FC1 and subtraction of the sample buffer difference curve from all other difference curves) as well as fitting of the data by the "bivalent analyte model” which assumes binding of one lgG2kappa by two adjacent molecules of biotin-protein G (dotted curves).
  • streptavidin was not only retained by biospecific binding to immobile desthiobiotin residues but also by physisorption, the synergy between the two being the likely reason for slow
  • the intention was to invert the nonspecific effect of the surface from attraction to repulsion of streptavidin.
  • the repulsion should be strong enough to help in the displacement of streptavidin by free biotin, while at the same time the repulsion should be small enough to allow for stable binding of streptavidin to the surface-linked desthiobiotin residues in absence of free biotin.
  • SAM self- assembled monolayer
  • OEG oligo(ethylene glycol) chains
  • SAMs which carry a dense brush of oligo(ethylene glycol) chains (OEG) are disclosed in Prime et al., 1991 and 1993, preferably with four ethylene oxide units or longer (Hahn et al. 2007, see molecule 4 in Fig. 2A).
  • the same high protein resistance is achieved if the SAM components lack long alkyl chains and consists only of PEG chains with terminal thiols (Nileback et al., 201 1 , see molecules 6 and 7 in Fig. 2A).
  • analogous SAMs with high protein resistance can be formed on H-terminated silicon, on acidic metal oxides, or on silanizable surfaces (glass, semiconductor oxide, metal oxide), respectively, if the thiol groups in 4, 6, and in homologous molecules are replaced by terminal vinyl groups (Booking et al., 2005; Yam et al., 2004), terminal
  • phosph(on)ate/catechol groups (Gnauck et al., 2007; Dalsin et al., 2005a and 2005b; Kotsokechagia et al., 2012), or terminal chloro/alkoxy-silane groups (Boozer et al., 2003) - or if dense PEG brushes are formed on pre-activated surfaces (Piehler et al. 2000).
  • Dense bottle-brush brushes, and dense coatings with multi-arm PEGs (Cha et al., 2004) and cross-linked hydrogels (Groll et al., 2005) serve the same purpose, whereby the PEG chains can be replaced by poly(2-methyl/ethyl-2-oxazolines) (POX, Fig. 2B) (Zhang, 2000) or poly(hydroxyethyl methacrylate) (pHEMA, Fig. 2B) (Konradi et al., 2007).
  • Bolduc et al. (2010) discovered that SAMs formed from the hydrophilic pentapeptide HHHDD (carrying a 3-mercaptopropionyl group on its N-terminus, see Fig.
  • anchoring groups on glass and metal oxides are larger than the tiny thiol groups which serve as anchors on gold surfaces.
  • longer hydrophilic polymer chains are typically grafted to the surface to create a protein-resistant monolayer on glass and related surfaces.
  • Biotin-SAMs on gold must not carry biotin residues on more than 30% of the oligoethylene glycol (OEG) chains, for otherwise streptavidin is prevented from binding (Jung et al., 2000). No such limitation was found on surfaces coated with long PEG chains (Biswas et al., 2014; Heyes et al., 2007). Obviously the surface covered by one long PEG chain (e.g., MW-2000, n ⁇ 50) is much larger than in SAMs with OEG chains, resulting in a significantly lower biotin density (Fig 3F - 3H). Moreover, the long, flexible polymer chains can easily adapt to the surface and the binding site geometry of bound streptavidin (Fig. 3F - 3H).
  • Fig. 3F - 3H When functionalized with desthiobiotin, the surfaces sketched in Fig. 3F - 3H are anticipated to show a similar functional behavior as the optimized SAM in Fig. 3B: Streptavidin can bind bivalently, thereby compensating for the lower binding strength of desthiobiotin as compared to biotin. At the same time, the limited extensibility of individual polymer chains (Kienberger et al. 2000) beyond the interface of the densely packed layer pulls bound streptavidin towards the protein-repelling polymer surface. On desthiobiotin-SAMs (Fig. 3B), this antagonism was found essential to ensure stable binding of streptavidin under normal measuring conditions and rapid removal upon rinsing with 200 mM biotin and/or 6 M GTC.
  • the principles of regenerative chip preparation can be adapted from gold surfaces to glass and oxide surfaces if the thin SAM is replaced by densely packed layers of longer linear polymers which form such dense brushes that streptavidin can only bind two polymer-linked desthiobiotins.
  • Branching sites depicted in Fig. 3G and Fig. 3H do not concern the linear polymer chains as such but their attachment to a linear or circular core structure which is then immobilized on the solid surface (Groll et al., 2005; Heyes et al., 2007; Huang et al., 2002) or prepared on the surface (Zhang, 2000). Branching of the core structure serves to force the linear chains into a maximally packed state, where the linear polymer chains are arranged in a quasi-parallel fashion and where the biotinylated ends of the hydrophilic chains can reach the layer surface, as needed for binding of streptavidin. Attachment of the linear polymer chains to a core polymer (Fig.
  • Fig. 3G or a circular core
  • Fig. 3H is only an option preferred by some workgroups to achieve high packing density but it is not intrinsically necessary (Fig. 3F), as shown by Piehler et al. (2000) and Biswas et al. (2014).
  • A is a moiety which provides for stable anchoring to a solid surface
  • n 0 or an integer of 1 to 22
  • X is selected from the group consisting of ether, thioether, ester, amide, urethane, urea, hydrazone, oxime, and acetal bond, as well as the triazole product formed from azide with alkyne,
  • L denotes a hydrophilic polymer (including hydrophilic peptides and peptoids)
  • Y is selected from the group consisting of an ether, thioether, ester, amide, urethane, urea, hydrazone, oxime, acetal bond, as well as the triazole product formed from azide with alkyne, and
  • D denotes desthiobiotin or a derivative thereof
  • the total linear chain of— (Ch n— X— L— Y— comprises at least 27 unbranched backbone atoms, with the proviso that D is not biotin.
  • the term "unbranched" refers to a linear and straight backbone atom chain without any branches. However, the backbone atoms may optionally bear a substituent or a sidechain. In contrast, in a branched polymer a substituent, e.g., a hydrogen atom on a monomer subunit is replaced by another covalently bonded chain of that polymer. Examples of suitable polymers are depicted in Fig. 2B. Not suitable for the present inventions are polymers with densely branched structure and a large number of end groups such as dendrimers. In some embodiments of the invention the linear unbranched polymer chains may be attached to a core polymer or a circular core.
  • the total linear unbranched chain of — (CH 2 )n— X— L— Y— comprises 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42 ,43, 44, 45, 50, 75, 100, 125, 150, 175 or up to 200 backbone atoms.
  • the total linear unbranched chain of — (CH 2 )n— X— L— Y— comprises 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42 ,43, 44, 45, 50, 75, 100, 125, 150, 175 or up to 200 backbone atoms.
  • the total linear chain of— (CH2)n— X— L— Y— comprises 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42 or 43 backbone atoms.
  • the total linear chain of— (CH2)n— X— L— Y— comprises 39, 40, 41 or 42 backbone atoms.
  • the total linear chain of— (CH2)n— X— L— Y— comprises 41 backbone atoms.
  • the total linear unbranched chain of — (CH 2 )n— X— L— Y— comprises 100, 125, 150, 175 or up to 200 backbone atoms.
  • first aspect A represents an organosulfur group, an organosilicon group, a fatty acid, a hydroxamic acid, a phosphonate or phosphate group, a catechol moiety (1 ,2-hydroxybenzene or 1 ,2,3-trihydroxybenzene), or a terminal vinyl group.
  • hydrophilic polymer refers to any suitable polymer which preferably avoids non-specific adsorption of proteins and nucleic acids.
  • Suitable hydrophilic polymer are for example, polyethylene glycol (PEG), polyoxazolines, polyethylene oxide) (PEO), polyvinyl pyrrolidone) (PVP), poly(methacrylic acid) (PMA), poly(acrylic acid) (PAA), poly(hydroxyethyl methacrylate) (pHEMA), polyvinyl alcohol) (PVA), peptides, peptoids, cellulose derivatives or a natural polymer such as d extra n.
  • PEG polyethylene glycol
  • PEO polyethylene oxide
  • PVP polyvinyl pyrrolidone
  • PMA poly(methacrylic acid)
  • PAA poly(acrylic acid)
  • pHEMA poly(hydroxyethyl methacrylate)
  • PVA polyvinyl alcohol
  • the hydrophilic polymer represents poly(ethylene glycol), polyacrylamide, polyvinyl alcohol), hydroxylethylcellulose (HEC), poly(/V-hydroxyethyl acrylamide) (PHEA), hydroxylpropyl methylcellulose (HPMC), poly(2-hydroxyethyl methacrylate) (pHEMA), polyvinyl pyrrolidone), poly(acrylic acid), dextran, hyaluronic acid, poly (2-methacryloyloxyethyl phosphorylcholine) (PMPC), or poly(2-methyl/ethyl-2-oxazoline) (POX), hydrophilic peptides and hydrophilic peptoids.
  • peptide refers to a peptide which comprises at least 3 to 10 amino acids which may be the same or different. Preferably the peptide comprises 4, 5, 6, 7 or 8 amino acids.
  • L represents polyethylene glycol
  • the polyethylene glycol comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 ethylene oxide moieties.
  • the polyethylene glycol comprises 5, 6, 7, 8 or 9 ethylene oxide moieties.
  • the polyethylene glycol comprises 6, 7 or 8 ethylene oxide moieties.
  • X denotes an amide group.
  • Y denotes an amide group.
  • the term "derivative of desthiobiotin” refers to a compound which mimics the binding properties of desthiobiotin as closely as possible, being homologous to desthiobiotin by differing from desthiobiotin only by insertion of one or two Ch groups in an existing C-C or N-H bond, or by insertion/deletion of up to 4 Ch groups in the linear hydrocarbon chain, or by exchange of the terminal carboxyl group by an amino alcohol or thiol group.
  • derivative of desthiobiotin does not encompass biotin.
  • the desthiobiotin derivative is of
  • R 1 , R 2 and R 3 are independently from one another H, or a substituted or C1-3 alkyl, C2-3-alkenyl, C2-3-alkynyl, and
  • R 4 is -(CH 2 ) P -R a , wherein
  • p is an integer from 1 to 8
  • R a is selected from the group consisting of -COOH, -CO-NH-NH2 (hydrazide), -CHO (aldehyde), -NH-NH2, -O-NH2, -NH 2 , -NH-CH3, -OH, -SH, or -N 3 or alkyne (as used in click chemistry).
  • the desthiobiotin derivative is of Formula II, wherein R 1 and R 2 are H, R 3 is -CH 3 , and R 4 is -(CH 2 )5-COOCH 3 .
  • the desthiobiotin derivative is of Formula II, wherein
  • R 2 is H
  • R 1 and R 3 are -CH 3
  • R 4 is -(CH 2 ) 5 -COOH.
  • the desthiobiotin derivative is of Formula II, wherein R 1 is -CH2CH3, R 2 is H, R 3 is -CH 3 , andR 4 is -(CH 2 ) 5 -COOH.
  • conjugate is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • Streptavidin was found to be stably bound on protein-resistant SAMs where a fraction of the OEG chains carries biotin residues, provided that the biotin-terminated alkanethiol (BAT) is longer than the matrix alkanethiol (MAT) by at least 7 backbone atoms (Jung et al., 2000, see molecules 4 and 5 in Fig. 2A). In case of wild-type streptavidin, the protein-repelling effect of the OEG layer was not very evident.
  • Streptavidin mutants were rapidly displaced by free biotin if the lateral density of biotin was low (BAT/MAT ⁇ 1/99). Interestingly, stable binding of these mutants on the mixed SAM was restored by using a higher lateral density of biotin (20- 30%).
  • Fig. 3A corresponds to a mixed SAM formed from molecules 1 and 2 (Fig. 2A).
  • the affinity is lower than on the analogous biotin-SAM (Knoll et al., 2000) but the linker segment between SAM and desthiobiotin is sufficiently long (12 atoms, indicated by ⁇ 12 in Fig.
  • Fig. 3B applies to the mixed SAM formed from components 8 and 9 (Fig. 2A) at a molar ratio of 80/20.
  • This mixed SAM is strictly analogous to our previously published biotin-SAM (Pollheimer et al., 2013), except that biotin has been replaced by desthiobiotin.
  • the linker between desthiobiotin and the SAM surface is shorter ( ⁇ 9, Fig. 2A), thus streptavidin is pulled towards the protein-repelling OEG layer.
  • bivalent binding would ensure stable binding of streptavidin in absence of biotin.
  • a protein-resistant SAM can also be formed by the hypothetical structure 3b which lacks one methylene group in the alkyl chain of structure 3 and has a linker length of 20 atoms.
  • the mixed SAM with 80% of component 8 (MAT) and 20% of component 9 (desthiobiotin-terminated alkanethiol, DBAT) turned out to be a preferred embodiment which provides for completely stable binding of streptavidin under all typical conditions of measurement and of "standard regeneration" (Fig. 1AB), while at the same time at least 99% of streptavidin plus the biotinylated sensor molecules are removed and replaced within few minutes when desired (Fig. 1 CD).
  • the present invention encompasses a sensor chip comprising a substrate and a self-assembled monolayer comprising a conjugate of Formula I as described above.
  • the present invention encompasses a sensor chip, wherein the substrate is selected from metal, semiconductor, metal oxides, semiconductor oxides, glass, metal and semiconductor surfaces primed with coordinating transition metal ions, hydrogen-terminated silicon, diamond, or plastic.
  • the present invention encompasses a sensor chip, wherein the substrate is selected from the group consisting of gold, silver, copper, lead, mercury, AI2O3, ⁇ 2, Nb2Os, Ta2Os, ITO, iron oxides, SnO2, S1O2, AgO, CuO.
  • the present invention encompasses a sensor chip, wherein the substrate is gold.
  • the present invention encompasses a sensor chip, wherein the self-assembled monolayer is a mixed self-assembled monolayer comprising a matrix component and a conjugate of Formula I as described above.
  • a further aspect of the invention relates to the sensor chip, wherein the matrix component (MAT) is a conjugate of Formula III,
  • the total linear chain of— (Ch jn— X— L comprises at least 20 backbone atoms.
  • the total linear chain of— (Ch n— X— L comprises 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30,0 31 , 32, 33, 34, 35, 36, or 37 backbone atoms.
  • the total linear chain of— (CH2)n— X— L comprises 25, 26, 27, 28, 29, 30,0 31 , 32, 33, 34, or 35 backbone atoms.
  • the total linear chain of— (Ch n— X— L comprises 32 backbone atoms.
  • A is a sulfur-containing group, preferably a thiol group.
  • X is an amide group.
  • the matrix component is selected from the group consisting of
  • a further aspect of the invention relates to the sensor chip as described above, wherein the matrix component is at least 4, 5, 6, 7, 8, 9, or 10 backbone atoms shorter compared to the compound of Formula I.
  • a further aspect of the invention relates to the sensor chip, wherein in said mixed self-assembled monolayer the ratio of compound of general Formula I to matrix component is in the range of about 3 to 50%, preferably in the range of about 5 und 40%, more preferred in the range of about 10 und 20%.
  • the mixed SAM is formed from
  • a further aspect of the invention relates to the sensor chip, wherein the self- assembled layer is functionalized by biospecific binding of a biomolecule.
  • the biosensor is functionalized for biospecific binding of biotin.
  • a further aspect of the invention relates to a sensor chip, wherein the functionalizing molecule is avidin, streptavidin, bradavidin, rhizavidin, or avidin-related proteins (AVRs) as well as mutants and/or hybrids and/or fusion proteins of said biotin- binding proteins.
  • the functionalizing molecule is avidin, streptavidin, bradavidin, rhizavidin, or avidin-related proteins (AVRs) as well as mutants and/or hybrids and/or fusion proteins of said biotin- binding proteins.
  • a further embodiment of the invention relates to a method for detecting an analyte molecule in a sample, comprising the steps of:
  • the biomolecule is streptavidin or avidin.
  • a sensor molecule according to the invention comprises a biotin moiety and a target moiety.
  • target moieties include synthetic molecules, nucleotides, nucleic acids, aptamers, peptide nucleic acids, peptides, proteins, enzymes, and antibodies.
  • antibody encompassed monoclonal antibodies (mAbs), immunoglobulin (Ig) or immunoglobulin class G (IgG), heavy-chain antibodies (HcAb's), or fragments thereof such as fragment-antigen binding (Fab), Fd, single- chain variable fragment (scFv), or engineered variants thereof such as for example Fv dimers (diabodies), Fv trimers (triabodies), Fv tetramers, or minibodies, single-domain antibodies like VH or VHH or V-NAR, and darpins.
  • the analyte molecule is complementary to the target moiety.
  • Complementary according to the invention refers to molecules which specifically can bind to the target moiety.
  • the corresponding antigen is used as analyte molecule.
  • a further embodiment of the invention relates to the method for detecting an analyte molecule, wherein the sensor molecule comprises an antibody and the analyte molecule is an antigen.
  • the sensor molecule comprises an antigen and the analyte molecule is an antibody.
  • a further embodiment of the invention relates to the method for detecting an analyte molecule, wherein the self-assembled monolayer is coated with a biomolecule such as for example, avidin or streptavidin and the sensor molecule is labelled with biotin, desthiobiotin, a peptide (e.g. Streptag), or an aptamer.
  • An aptamer according to the invention refers to an oligonucleotide or peptide that binds a specific target molecule. More specifically, aptamers can be classified as DNA, RNA or XNA aptamers usually consisting of short strands of oligonucleotides and peptide aptamers. Peptide aptamers consist of a short variable peptide domain, attached at both ends to a protein scaffold.
  • the analyte molecule may be tagged or labeled for detection such that a detectable signal is produced.
  • the detectable signal is produced by any of the tags or labels known in the art.
  • tags or labels refers to any substance attachable to an analyte molecule, in which the substance is detectable by a detection method.
  • labels applicable to this invention include but are not limited to luminescent molecules, chemiluminescent molecules, fluorochromes, fluorescent quenching agents, colored molecules, radioisotopes, scintillants, massive labels (for detection via mass changes), biotin, avidin,
  • streptavidin protein A, protein G, antibodies or fragments thereof, Grb2, polyhistidine, Ni 2+ complexes, Flag tags, myc tags, heavy metals, enzymes, alkaline phosphatase, peroxidase, luciferase, electron donors/acceptors (e.g., methylviologen or methylene blue), acridinium esters, labels for electrochemiluminescence (e.g., rubidium
  • a method for regenerating a sensor chip comprises the steps of: (a) washing the sensor chip with a chaotropic agent,
  • a further aspect of the invention encompasses the method as described above, wherein the chaotropic agent is urea, guanidinium chloride, or guanidinium thiocyanate.
  • the chaotropic agent may be used in combination with HCI or SDS.
  • the chaotropic agent is guanidinium thiocyanate.
  • the chaotropic agent is used in the range of 6 to 8 M, preferably 6 M.
  • a further aspect of the invention encompasses the method for regenerating a sensor chip, wherein the proteolytic enzyme is pepsin.
  • a further aspect of the invention encompasses the method for regenerating a sensor chip, wherein pepsin is used at low pH, preferably about pH 2 and in an amount of about 0.5 - 5 mg/ml, preferably 1 - 4 mg/ml, more preferred of about 2 mg/ml.
  • a further aspect of the invention encompasses the method for regenerating a sensor chip, wherein the ionic surfactant is an anionic surfactant, preferably sodium dodecyl sulfate.
  • a further aspect of the invention encompasses the method for regenerating a sensor chip, wherein the sensor chip is pretreated with 100 - 500 mM biotin, preferably with 150 - 300 mM, more preferred with about 200 mM.
  • a further aspect of the invention encompasses the method for regenerating a sensor chip, wherein at least 95%, preferably 97%, more preferably at least 98% of the self-assembled monolayer is regenerated.
  • Regenerated according to the inventions means that the fraction of the non-covalently bound complex comprising the
  • the biomolecule, the sensor molecule and the analyte molecule is removed by the regeneration procedure and the sensor chip can be reused for further cycles of sensor molecule immobilization and analyte detection.
  • the self-assembled monolayer of the sensor chip remains functional.
  • a further aspect of the invention encompasses the method for regenerating a sensor chip, wherein said sensor chip is reused and again regenerated.
  • the sensor chip is regenerated at least 3 times, at least 10 times or at least 20 times. Due to the effective method for regenerating the sensor chip is still usable after several cycles of regeneration. Examples
  • Components 9 and 10 were synthesized by reacting the N- hydroxysuccinimide ester of 16-bromohexadecanoic acid with the appropriate NH2- PEG-NH-Boc (Polypure, Oslo, Norway) in chloroform and N,N-diisopropyl-N- ethylamine (DIEA). The product was purified by washing the chloroform solution with phosphoric acid, sodium carbonate, saturated NaCI, and by chromatography on Sephadex LH-20 (in chloroform).
  • DIEA N,N-diisopropyl-N- ethylamine
  • the Boc group was removed with trifluoroacetic acid (TFA) in dichloromethane and the N-hydroxysuccinimide ester of desthiobiotin was coupled to the deprotected amino group in DMF and DIEA.
  • TFA trifluoroacetic acid
  • Excess of desthiobiotin NHS-ester was hydrolyzed in pyridine/water (9/1 ) at 30-35°C for 2 h to allow for easier removal of NHS and desthiobiotin by washing a chloroform solution of the crude product with 0.1 M phosphoric acid, with 10% sodium carbonate, and with saturated NaCI solution.
  • the bromine atom was replaced by thioacetate in DMF/potassium thioacetate, the product was purified by chloroform/water extraction, and the acetylthio groups were hydrolyzed with K-tert-butoxide in methanol under strict exclusion of oxygen up to the moment of acidification with TFA.
  • the reaction mixture was diluted with chloroform and washed with aqueous NaCI solution (-2.5 M), yielding the pure product in the chloroform layer.
  • the reaction conditions and purification methods extraction, gel filtration on Sephadex LH20 in chloroform) were closely similar as in the synthesis of the analogous biotin component (Pollheimer et al., 2013).
  • the matrix component 8 (Fig. 2A, synthesized as described in Pollheimer et al., 2013) and the desthiobiotin components (9 or 10) were mixed from chloroform stock solutions at the desired molar ratio (80/20, except for Example No. 6 where the molar ratio was 99/1 ), dried, redissolved in THF, and treated with zinc/acetic acid, as described for the analogous mixed biotin-SAM components (Pollheimer et al., 2013).
  • This step ensured a statistical distribution of the symmetric and mixed disulfides of 8 and 9 (or 8 and 10) after reoxidation in air and greatly improved the functional properties of the mixed SAM (Pollheimer et al, 2013).
  • Pretreatment of gold and SAM formation was performed as described for the analogous biotin-SAM (Pollheimer et al., 2013).
  • a 400 mM stock solution of free biotin was prepared by addition of Tris base to a final pH of 8.0.
  • Mixed SAMs with 20% desthiobiotin component 9 were prepared as described in Example No. 2.
  • Streptavidin was bound in both flow cells of the SPR biosensor (BIAcore X), and biotinylated goat IgG (biotin-lgG) was immobilized on top of streptavidin in flow cell 2 (FC2).
  • Free biotin was injected at 0.1 mM, 1 mM, 10 mM concentration, as well as at 100 mM, 200 mM, and 400 mM concentration.
  • concentrations might have a two-fold effect: (i) the well-known competition for the biotin-binding sites of streptavidin which are occupied by the desthiobiotin residues of the chip and by the biotin residues of biotin-lgG, and (ii) a hitherto unreported activity of deprotonated biotin as a very strong denaturant. The latter seemed likely because of the amphiphilic nature of deprotonated biotin, as well as because of the urea segment of biotin.
  • TCEP -free GTC was much preferred because TCEP is sensitive to oxidation by air, thus (immediately before the injection) GTC must be mixed with acidic TCEP hydrochloride and with the appropriate amount of a base, and this step cannot be performed by standard autosamplers.
  • GTC had almost no effect on the double layer of streptavidin and statistically biotinylated BSA but these proteins were easily removed in the subsequent injection of pepsin (dashed trace in Fig. 8E).
  • pepsin acts by cleavage of the biotinylated protein (see sketch in Fig. 8D).
  • Example 6 Mixed SAM with a longer linker between the SAM surface
  • the mixed SAM of components 8 and 9 in an 80/20 ratio provides for an optimal situation where strain and repulsion of streptavidin facilitate its removal by GTC, pepsin, and biotin.
  • bivalent binding is able to ensure stable binding of streptavidin in absence of these reagents, in spite of strain and protein repulsion.
  • Fig. 12 demonstrates that nonspecific DNA adsorption to sensor chip-bound streptavidin is below the detection limit.
  • the figure shows only the essential part of the whole experiment which followed the same protocol as in Taskinen et al. (2014).
  • a streptavidin monolayer was formed in FC2 and functionalized with biotin-labeled DNA probe (a 30 mer with the same sequence as in Taskinen et al. (2014)).
  • a streptavidin monolayer was formed in FC1 and biotin-BSA was run over both flow cells (the first injection plotted in Fig. 12). After injection of unlabeled BSA, the same DNA strand as before was injected, except that it lacked the biotin label. No binding was detected in any of the two flow cells, proving absence of nonspecific adsorption.
  • Immobilized biotin-protein G and soluble human lgG2i constitute a good test system for Biological Interaction Analysis in a biosensor (Pollheimer et al., 2013;

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Abstract

La présente invention concerne le domaine des capteurs, en particulier une puce de capteur pour détecter un analyte dans un échantillon. La puce de capteur comprend une couche de substrat et un ou plusieurs conjugués organiques liés au substrat sous la forme d'une monocouche auto-assemblée (SAM). La présente invention concerne également un nouveau conjugué utilisé pour former une monocouche auto-assemblée (SAM). Des procédés de régénération de la puce de capteur permettant son utilisation répétée sont en outre décrits.
PCT/EP2016/069164 2015-08-14 2016-08-11 Biocapteur à régénération WO2017029194A1 (fr)

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WO2005080989A1 (fr) * 2004-02-13 2005-09-01 Molecular Probes, Inc. Capteurs de reconnaissance de la biotine et essais a fort debit
EP2259068A2 (fr) * 2003-01-16 2010-12-08 caprotec bioanalytics GmbH Composés de capture et procédés d'analyse protéomique
WO2012058635A1 (fr) * 2010-10-29 2012-05-03 Life Technologies Corporation Dérivés de biotine
WO2015023059A1 (fr) * 2013-08-13 2015-02-19 고려대학교 산학협력단 Procédé de préparation de nanostructure métallique sur la base de biomolécules

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DE10144251A1 (de) * 2001-08-31 2003-03-27 Fraunhofer Ges Forschung Vorrichtung für die gerichtete Immobilisierung von Proteinen
EP2259068A2 (fr) * 2003-01-16 2010-12-08 caprotec bioanalytics GmbH Composés de capture et procédés d'analyse protéomique
WO2005080989A1 (fr) * 2004-02-13 2005-09-01 Molecular Probes, Inc. Capteurs de reconnaissance de la biotine et essais a fort debit
WO2012058635A1 (fr) * 2010-10-29 2012-05-03 Life Technologies Corporation Dérivés de biotine
WO2015023059A1 (fr) * 2013-08-13 2015-02-19 고려대학교 산학협력단 Procédé de préparation de nanostructure métallique sur la base de biomolécules

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HYUN C. YOON ET AL: "Reversible Association/Dissociation Reaction of Avidin on the Dendrimer Monolayer Functionalized with a Biotin Analogue for a Regenerable Affinity-Sensing Surface", LANGMUIR, vol. 17, no. 4, 1 February 2001 (2001-02-01), NEW YORK, NY; US, pages 1234 - 1239, XP055241992, ISSN: 0743-7463, DOI: 10.1021/la001373g *
JAMES D HIRSCH ET AL: "Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation", ANALYTICAL BIOCHEMISTRY, ACADEMIC PRESS INC, NEW YORK, vol. 308, no. 2, 15 September 2002 (2002-09-15), pages 343 - 357, XP002462135, ISSN: 0003-2697, [retrieved on 20020916], DOI: 10.1016/S0003-2697(02)00201-4 *
PHILIPP POLLHEIMER ET AL: "Reversible Biofunctionalization of Surfaces with a Switchable Mutant of Avidin", BIOCONJUGATE CHEMISTRY., vol. 24, no. 10, 16 October 2013 (2013-10-16), US, pages 1656 - 1668, XP055241869, ISSN: 1043-1802, DOI: 10.1021/bc400087e *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101868917B1 (ko) * 2017-02-06 2018-06-19 울산과학기술원 단백질 표지화용 페놀 화합물 및 이의 제조방법

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