WO2014020293A1 - Dosage - Google Patents

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Publication number
WO2014020293A1
WO2014020293A1 PCT/GB2012/051852 GB2012051852W WO2014020293A1 WO 2014020293 A1 WO2014020293 A1 WO 2014020293A1 GB 2012051852 W GB2012051852 W GB 2012051852W WO 2014020293 A1 WO2014020293 A1 WO 2014020293A1
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WIPO (PCT)
Prior art keywords
moiety
signal
binding
analyte
reducing agent
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PCT/GB2012/051852
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English (en)
Inventor
Roberto De La Rica Quesada
Molly Morag Stevens
Laura RODRÍGUEZ-LORENZO
Luis LIZ-MARZÁN
Original Assignee
Imperial Innovations Limited
Universidade De Vigo
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Application filed by Imperial Innovations Limited, Universidade De Vigo filed Critical Imperial Innovations Limited
Priority to PCT/GB2012/051852 priority Critical patent/WO2014020293A1/fr
Publication of WO2014020293A1 publication Critical patent/WO2014020293A1/fr

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    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate

Definitions

  • the present invention relates to an assay and a kit for determining the presence of an analyte in a liquid sample.
  • Lowering the limit of detection is a fundamental aspect in the design of sensors important to food safety regulations, 1"2 environmental policies 3"5 and the diagnosis of severe diseases. 6"10
  • conventional transducers generate a signal that is directly proportional to the concentration of the target molecule.
  • the presence of chemical species at ultralow concentrations results in tiny variations in the physical properties of the sensor and these are difficult to detect with confidence.
  • Metal nanoparticles can be used as building blocks for the fabrication of biosensors when their localized surface plasmon resonance (LSPR) shifts in response to a biorecognition event. 11 In these detection platforms, the largest variations in the LSPR are observed when another metallic nanostructure interacts in close proximity with the nanoparticle.
  • LSPR localized surface plasmon resonance
  • the present invention provides a method of determining the presence of an analyte in a liquid sample, comprising:
  • the concentration of the reducing agent-generating moiety around the signal moiety is directly related to the concentration of the analyte. This is achieved by binding the reducing agent-generating moiety to the reaction conjugate.
  • the reducing agent-generating moiety may be linked to a second binding moiety which binds the reaction conjugate directly, and preferably binds the analyte in the reaction conjugate.
  • the second binding moiety may bind the reaction conjugate indirectly via one or more intermediate binding moieties, at least one of which binds the reaction conjugate, preferably the analyte.
  • the method preferably includes the step of removing analyte which is not bound to the first binding moiety prior to binding any further binding moieties to the analyte in the reaction conjugate.
  • reducing agent is generated which reduces the metal ions.
  • the reduction of metal ions to metal atoms causes either the formation of a homogeneous metal coating on the signal moiety or the formation of free-standing metal nanocrystals.
  • the short supply of reducing agent dictates slow crystal growth conditions. This favours the growth of a homogeneous metal coating on the signal moiety acting as a seeding point, 16 which yields a change in the signal yielded by the signal moiety. 17,18
  • the concentration of the reducing agent- generating moiety is high (due to a high concentration of analyte)
  • the abundant supply of reducing agent favours nucleation instead of epitaxial growth and free-standing metal nanocrystals are obtained. 16 Consequently, less metal is deposited on the signal moiety and the signal change is less than in the presence of reducing agent-generating moiety at low concentrations.
  • the reducing agent-generating moiety will not be localized to the signal moiety. Therefore the metal ions will not be reduced and there will be no change in the signal generated by the signal moiety because no coating will form on the signal moiety.
  • the presence of the target analyte at low concentrations yields a larger change in the signal yielded by the signal moiety via the formation of a metal coating.
  • concentration and signal herein referred to as "inverse sensitivity”
  • inverse sensitivity makes this approach highly suitable for the fabrication of ultrasensitive sensors. This is because the change in signal yielded by the signal moiety when the analyte is present at very low concentrations is the highest, making it easy to differentiate it from when there is no change in signal due to an absence of the analyte.
  • the assay can be used to determine the presence or concentration of an analyte even when the analyte is present in a very low concentration and, for example, in a complex matrix such as bodily fluids.
  • the assay of the present invention can be used to detect analyte at a concentration of as low as about 1 x 10 "12 g ml "1 or less, about 1 x 10 "13 g ml “1 or less, about 1 x 10 "14 g ml “1 or less, about 1 x10 "15 g ml “1 or less, about 1 x 10 "16 g ml “1 or less , about 1 x 10 "17 g ml “1 or less and most preferably about 1 x10 "18 g ml “1 or less (which is close to the single-molecule level).
  • the first binding moiety is contacted with a liquid sample.
  • analyte binds to the first binding moiety to form a reaction conjugate.
  • the liquid sample may be incubated with the first binding moiety for a period of time, for example about 0.5 to about 5 hours inclusive, about 1 hour to about 4 hours inclusive, about 1.5 hours to about 3 hours inclusive and preferably about 2 hours.
  • the reaction conjugate may then be washed, for example by centrifugation, and then may be re-dispersed in buffer.
  • a detection conjugate is formed by binding the reaction conjugate to a reducing agent-generating moiety.
  • the reducing agent- generating moiety is linked to a second binding moiety which binds the reaction conjugate.
  • unbound second binding moiety linked to the reducing agent-generating moiety may be washed away.
  • the second binding moiety may bind the reaction conjugate indirectly via one or more intermediate binding moieties, at least one of which binds the reaction conjugate.
  • any unbound intermediate binding moieties may be washed away prior to the second binding moiety binding the one or more intermediate binding moieties.
  • the detection conjugate is then contacted with a substrate for the reducing agent-generating moiety. This causes a reducing agent to be generated.
  • the reaction mixture is contacted with metal ions which are reduced by the reducing agent to metal atoms.
  • metal ions which are reduced by the reducing agent to metal atoms.
  • the coating causes a change in the signal yielded by the signal moiety. The presence of an analyte can therefore be detected by determining whether there has been a change in the signal yielded by the signal moiety, thus providing a qualitative test.
  • the method can readily be transformed into a quantitative test whereby the concentration of the analyte in the sample can be determined.
  • concentration of the analyte in the sample can be determined.
  • This may be achieved using a calibration curve such as the one shown in Figure 2 of the accompanying drawings.
  • the change in signal is larger when the analyte is less concentrated (i.e. the assay is inversely sensitive) and there is no change in signal when analyte is not present, the invention allows one to differentiate samples containing no analyte from samples containing very low levels of analyte with a high degree of confidence.
  • the sample may be tested and the signal interpolated in calibration curves similar to those provided in Figure 2 of the accompanying drawings.
  • the sample may be serially diluted 1 :10 several times.
  • the signal moiety which is linked (covalently or non-covalently) to the first binding moiety can be, for example, a plasmonic sensor, a fluorescent moiety or an electrochemical sensor.
  • a plasmonic sensor is preferably a coinage metal nanostructure that shows the localized surface plasmon resonance effect.
  • Suitable plasmonic sensors include nanoparticles, for example spherical silver nanoparticles, gold nanoparticles, silver prisms, gold nanorods, gold nanoflowers and gold nanostars as well as other nanostructures such as core-shell silica-gold colloids.
  • the plasmonic sensor is a gold nanostar.
  • the signal to be measured is preferably the Localized Surface Plasmon Resonance (LSPR) of the plasmonic sensor.
  • LSPR Localized Surface Plasmon Resonance
  • metal coating is deposited on the plasmonic sensor, this yields a shift in the LSPR of the plasmonic sensor. 17 18 This shift in LSPR indicates the presence of the analyte.
  • the shift in LSPR is caused by the hybridization of the dielectric constants of the plasmonic sensor and the metal coating. For example, when a silver coating is deposited on a gold nanostar, the LSPR of the gold nanostar can be shifted by as much as 150 nm.
  • the signal moiety is a fluorescent moiety.
  • the fluorescent moiety may be a fluorescent particle such as a fluorescent nanoparticle.
  • the fluorescent moiety is preferably made from a fluorescent material.
  • the fluorescent material is a semiconductor material, such as, for example, CdS, CdSe, CdTe, ZnS, AgS, PbS, ZnO, Ti0 2 as well as core-shell structures of two different materials (e.g a CdSe core surrounded by a ZnS shell).
  • the fluorescent moiety can also be an upconverting nanoparticle such as a lanthanide- doped nanoparticle.
  • the signal to be measured is preferably the fluorescence of the fluorescent moiety. When metal coating is deposited on the fluorescent moiety this quenches the fluorescence of the fluorescent moiety and a change in fluorescence indicates the presence of the analyte.
  • the signal moiety is an electrochemical sensor.
  • the electrochemical sensor may be an electrode such as a noble metal (e.g. gold, platinum) electrode, a mercury drop electrode or a carbon-based electrode.
  • a potentiometric sensor for example a field-effect transistor or a glass electrode may be used.
  • the signal to be measured is preferably the electrochemical potential of the electrochemical sensor, and a change in electrochemical potential indicates the presence of the analyte.
  • the signal may be the reduction potential.
  • the first, second and, where present, the one or more intermediate binding moieties can each be any moiety which binds a target. Such binding may be specific.
  • the target is the analyte.
  • the target is the reaction conjugate, preferably the analyte in the reaction conjugate or the one or more intermediate binding moieties where these are used.
  • the one or more intermediate binding moieties at least one of these binds the reaction conjugate.
  • Further intermediate binding moieties may be added in series, each binding to the intermediate binding moiety preceding it with the second binding moiety binding the final intermediate binding moiety added.
  • the binding moiety may be naturally derived or wholly or partially synthetically produced.
  • the binding moiety and target together form a pair of binding partners.
  • One member of the pair has an area on its surface, which may be a protrusion, cavity or a particular chemical function which specifically binds to and is therefore complementary to a particular spatial and polar organisation of the other member of the pair.
  • the members of the pair have the property of binding specifically to each other.
  • types of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, DNA-DNA, receptor-ligand and enzyme-substrate.
  • the present invention is generally concerned with antigen-antibody type reactions.
  • preferred binding moieties of the invention are antibodies.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, whether natural or partly or wholly synthetically produced.
  • the term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. These can be derived from natural sources, or they may be partly or wholly synthetically produced.
  • antibodies are the immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies.
  • Antibodies may be polyclonal or monoclonal. A monoclonal antibody may be referred to herein as "mab".
  • antibody should be construed as covering any specific binding member or substance having a binding domain with the required specificity.
  • this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, humanised antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023.
  • a humanised antibody may be a modified antibody having the variable regions of a non-human, e.g. murine, antibody and the constant region of a human antibody. Methods for making humanised antibodies are described in, for example, US Patent No. 5225539.
  • binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S.
  • Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g. by a peptide linker) but unable to associate with each other to form an antigen binding site: antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804).
  • bispecific antibodies may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Hollinger & Winter, Current Opinion Biotechnol. 4:446-449 (1993)), e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain "Janusins" described in Traunecker et a/., EMBO Journal 10:3655-3659 (1991 ).
  • Bispecific diabodies as opposed to bispecific whole antibodies, may also be useful because they can be readily constructed and expressed in E. coli.
  • Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (W094/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.
  • an “antigen binding domain” is the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope.
  • An antigen binding domain may be provided by one or more antibody variable domains.
  • An antigen binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • Specific is generally used to refer to the situation in which the binding moiety will not show any significant binding to molecules other than its target(s), and, e.g., has less than about 30%, preferably 20%, 10%, or 1 % cross-reactivity with any other molecule.
  • the term is also applicable where e.g. an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case, the binding moiety carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.
  • the first, second and one or more intermediate binding moieties do not necessarily have to be the same type of binding moiety, although they may be in some embodiments.
  • the first binding moiety is a polyclonal antibody and the second binding moiety is a monoclonal antibody. If one or more intermediate binding moieties are used, it is preferred if the first binding moiety is a polyclonal antibody and at least one intermediate binding moiety which binds the analyte is a monoclonal antibody.
  • the reducing agent-generating moiety can be any moiety that, when supplied with its substrate, is capable of generating a reducing agent that reduces metal ions.
  • Suitable reducing agents include hydrogen peroxide (H2O2), para-nitrophenol and the reduced form of nicotinamide adenine dinucleotide (NADH 2 ).
  • H2O2 hydrogen peroxide
  • NADH 2 nicotinamide adenine dinucleotide
  • the reducing agent-generating moiety is an enzyme.
  • suitable enzymes include glucose oxidase (GOx) which generates H 2 0 2 in the presence of glucose, alkaline phosphatase which generates para-nitrophenol in the presence of para-nitrophenylphosphate and alcohol dehydrogenase which generates NADH 2 in the presence of ethanol, although the skilled person will be aware of other enzymes which can perform the function of generating a reducing agent that reduces metal ions.
  • the enzyme is GOx.
  • the metal ions are reduced to metal atoms which may form a metal coating on the signal moiety or free-standing metal nanocrystals.
  • Any metal ions can be used in the invention provided that the deposition of the metal atom formed by reduction of the metal ion results in a change in size, morphology, state of aggregation, fluorescence emission or electrochemical potential of the signal moiety, causing a change in the signal yielded by the signal moiety.
  • gold, silver, copper, cobalt, nickel, lead or mercury ions could be used.
  • metal ions may be generated using an ionic compound by adding this to the reaction mixture.
  • a reagent which reacts with the ionic compound to form metal ions may be used.
  • silver nitrate and ammonia are added to generate silver ions.
  • the method described herein can be adapted to detect any analyte of interest.
  • the analyte of interest may be chosen from any species capable of undergoing a binding event with the first binding moiety.
  • the analyte may be a biomarker for a disease, a nucleic acid, a pollutant, an allergen, a contaminant or an antigen derived from a pathogen.
  • the assay is used to detect a cancer biomarker such as prostate-specific antigen (PSA).
  • PSA prostate-specific antigen
  • liquid sample includes any liquid which may contain an analyte of interest. Any raw sample may be pre-treated if necessary to obtain and/or release the analyte of interest and the treatment process may involve treating a solid sample to yield a liquid sample containing analyte of interest.
  • liquid sample includes bodily liquids that can be obtained from a mammalian body, including, for example, blood, plasma, urine, lymph, gastric juices, bile, serum, saliva, sweat, interstitial fluid and spinal and brain fluids. Furthermore, the bodily liquids may be either processed (e.g., serum) or unprocessed. Depending upon the analyte of interest, other fluid and liquid samples may be contemplated such as ones from industrial, environmental or agricultural sources.
  • the present invention provides a kit for determining the presence of an analyte in a liquid sample, comprising: (i) a signal moiety which yields a signal linked to a first binding moiety which specifically binds the analyte to form a reaction conjugate;
  • binding moieties for binding the reducing agent-generating moiety to the reaction conjugate.
  • the reducing agent-generating moiety is linked to a second binding moiety that is adapted to bind the reaction conjugate directly, preferably by binding the analyte.
  • the reducing agent-generating moiety is linked to a second binding moiety that binds the reaction conjugate indirectly, preferably by binding one or more intermediate binding moieties, at least one of which binds the reaction conjugate, preferably the analyte.
  • the kit may comprise one or more intermediate binding moieties.
  • the kit may also comprise one or more of the following: calibration data e.g.
  • a calibration curve showing analyte concentration in relation to change in signal for example the shift in LSPR when a plasmonic sensor is used
  • one or more washing buffers for example the shift in LSPR when a plasmonic sensor is used
  • one or more working buffers for example, one or more substrates for the reducing agent-generating moiety
  • one or more ionic compounds used to generate metal ions such as, for example, silver nitrate
  • one or more reactants that react with ionic compounds to generate metal ions such as for example, ammonia.
  • Preferred features of each aspect of the invention are as for each other aspect mutatis mutandis.
  • the prior art documents mentioned herein are incorporated to the fullest extent permitted by Law.
  • Figure 1 shows one embodiment of a signal generation mechanism via enzyme-guided crystal growth.
  • Glucose oxidase generates hydrogen peroxide, which reduces silver ions to grow a silver coating around plasmonic nanosensors (gold nanostars); i) at low concentrations of GOx, the nucleation rate is slow, which favours the growth of a conformal silver coating that induces a large blue shift in the LSPR of the nanosensors; ii) when GOx is present at high concentrations, the fast crystal growth conditions stimulate the nucleation of Ag nanocrystals and less silver is deposited on the nanosensors, therefore generating a smaller variation of the LSPR.
  • this signal generation step induces inverse sensitivity because condition (i) is fulfilled at low concentrations of analyte.
  • FAD and FADH 2 are the oxidized and reduced forms of flavin adenine dinucleotide.
  • Figure 2 relates to an immunoassay for the ultrasensitive detection of PSA with GOx-labeled antibodies.
  • Figure 3 shows SERS spectra of: (a) P VP-stabilized gold nanostars, (b) after modification with glutaraldehyde to yield 2 (see reaction scheme in methods section), (c) after covalent attachment of proteins (in this example GOx) to obtain 3 (see reaction scheme in methods section).
  • Inset amplification of the spectral region between 600 and 200 cm "1 .
  • Figure 4 shows intensity of the band at 288 cm “1 (a) and 383 cm “1 (b) with respect to the concentration of GOx when the protein immobilization step is performed in the presence or in the absence of glutaraldehyde.
  • Figure 5 shows Vis-NIR spectra of protein-modified gold nanostars in water ( ⁇ ) and in 0.3 M NaCI (o).
  • Figure 6 shows XEDS analysis of free-standing silver nanoparticles found in the solution containing 10 "14 g mL 1 GOx after the signal amplification step
  • Figure 7 shows XEDS analysis of gold nanostars that were modified with no GOx after the signal amplification step.
  • Figure 8 shows XEDS analysis of gold nanostars modified with 10 ⁇ 20 g mL "1 GOx after the signal amplification step.
  • Figure 9 shows XEDS analysis of gold nanostars modified with 10 "14 g mL “1 GOx after the signal amplification step.
  • Figure 10 shows Vis-NIR spectra of gold nanostars after immunodetection of PSA diluted in PBS to the final concentration of 0 g mL “1 , 10 "13 g mL “1 , 10 "14 g mL “1 , 10 "15 g mL “1 , 10 "16 g mL “1 , 10 "17 g mL “1 and 10 "18 g mL “1 .
  • Figure 11 shows Vis-NIR spectra of gold nanostars after immunodetection of PSA diluted in whole serum to the final concentration of 0 g mL “1 , 10 "14 g mL “1 , 10 "15 g-mL “1 , 10 "16 g mL “1 , 10 “17 g mL “1 and 10 "18 g mL “1 .
  • Figure 12 shows inverse sensitivity in plasmonic nanosensors.
  • this figure shows a) TEM image of gold nanostars (scale bar: 50 nm); b) Visible/near infrared spectra of the nanosensors, modified with 10 "14 g mL “1 GOx, 10 "20 g mL “1 GOx and without GOx after the signal generation step; c) blue shift of the LSPR absorbance band (AAmax) as a function of the concentration of GOx in the immobilization solution when the signal generation step is performed in the absence or in the presence of the enzyme substrate glucose (semilogarithmic scale).
  • this figure shows TEM pictures after the signal generation step when gold nanostars were modified with a) 10 "20 g mL “1 GOx, and b) 10 "14 g mL “1 GOx (scale bar: 50 nm); STEM image (c) and XEDS map (d) showing the distribution of gold and silver around nanostars modified with 10 "14 g mL "1 GOx (scale bar: 20 nm).
  • XEDS spectra are shown in Figures 6-9.
  • SERS surface-enhanced Raman spectroscopy
  • the inelastic scattered radiation was collected with a Renishaw Invia Reflex system, equipped with a two-dimensional Peltier charge-coupled device (CCD) detector and a confocal Leica microscope.
  • the spectrograph has 1200 g/mm grating with additional band-pass filter optics. Samples were excited with a 785 nm (diode) laser line. Samples for SERS were prepared by drop-casting 10 pL of the resulting dispersions on glass slides.
  • Spectra were collected by focusing the laser line onto the sample by using a 50 ⁇ objective (N.A. 0.75), providing a spatial resolution of about 1 pm 2 , with accumulation times of 10 s.
  • the covalent coupling of the protein to the nanostar can be inferred through the spectral changes observed in their corresponding SERS spectra (see Figure 3).
  • the SERS spectrum completely changes its vibrational profile. This is typical of the generation of high SERS cross-section moieties in the low cross-section aliphatic polymer.
  • the spectrum b clearly shows characteristic bands due to the ring including ring stretching and CCH in plane bendings (region from 1400 to 1600 cm “1 ) and ring breathings (996 and 1070 cm “1 ).
  • the SERS spectrum changes slightly.
  • the vibrational variations are accumulated in those regions described and are mainly due to the change in the orientation of the aromatic ring with respect to the plasmonic surface because of the steric hindrance induced by the protein.
  • the vibrational change can be completely explained in full agreement with the surface selection rules.
  • additional evidence of the coupling is also shown in the change of the relative intensity of the bands between 1500 and 1600 cm “1 .
  • the new profile shows ring stretching contributions together with the characteristic amide bands of proteins, especially those of the amide I at around 1650 cm "1 28
  • the intensity of the bands at 288 and 383 cm “1 was measured in the presence or in the absence of the glutaraldehyde linker.
  • the band at 288 cm “1 corresponds to the CH 2 rocking characteristic of amines; the band at 383 cm “1 was selected for its higher intensity.
  • the intensity of the bands increases as the concentration of GOx in the immobilization solution increases when the process is performed in the presence of glutaraldehyde as described above. However, in the absence of glutaraldehyde, only a small increase is registered, which is attributed to minor physisorption of the protein on the nanosensors.
  • PVP poly(vinylpyrrolidone)
  • the prepared nanostars have an average total size of 60 ⁇ 8 nm. Protocols for covalent attachment of biomolecules to PVP-stabilized gold nanostars can be found above.
  • the protein- modified nanosensors are stable in solutions containing highly concentrated electrolytes as discussed below.
  • H 2 O 2 was initiated by adding glucose (100 mM) to GOx- modified nanostars in MES buffer (10 mM, pH 5.9) for 1 h. Subsequently, AgN0 3 (0.1 mM) and NH 3 (40 mM) were added to trigger the reduction of silver ions on the gold nanosensors. Spectral changes were measured with a Jasco V-670 UV-vis-NIR spectrophotometer after 2 h. High resolution (HRTEM) and scanning transmission electron microscopy (STEM) images were obtained with a JEOL JEM 2010 FEG-TEM microscope operating at an acceleration voltage of 200 kV.
  • HRTEM high resolution
  • STEM scanning transmission electron microscopy
  • XEDS X-ray energy dispersive spectra
  • HAADF high angle annular dark field
  • Mapping was performed with a 0.7 nm probe size, and the acquisition time was limited to 60 s to avoid sample drift.
  • the corresponding quantitative EDS measurements were performed using an INCA EDS microanalysis system from Oxford Instruments. Determination of the relative amount of silver in the coatings was carried by standardless analysis using the Cliff-Lorimer correction with absorbance. Calculations were performed on three different sites on the grid.
  • the conjugation of GOx to anti-mouse IgG was performed by converting amino groups in the antibody to thiolate groups with 2-iminothiolane followed by conjugation with GOx via the heterofunctional linker sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC). 29 To 5 mg of glucose oxidase in 1 mL of PBS buffer pH 7.6, 200 ⁇ of sulfo-SMCC in water (5 mg/mL) was added two times at 30 min intervals. The reaction mixture was incubated for 1 hour at room temperature with periodic mixing.
  • the maleimide-activated GOx was purified with a P10 desalting column using PBS as eluate. Protein-rich fractions were identified by their characteristic absorbance peak at 280 nm.
  • polyclonal anti-mouse IgG developed in goat (Sigma) was dissolved to a concentration of 1 mg/mL in PBS. Then, 100 ⁇ of 1 .5 mg/mL 2-iminothiolane solution was reacted with the antibody for 1 hour at room temperature. The resulting thiolated antibody was purified with the desalting column using PBS as eluate; 1 mL fractions were collected and monitored for protein at 280 nm.
  • the fractions containing antibody were pooled and immediately mixed with the maleimide-modified glucose oxidase. After overnight incubation at 4°C, the GOx-modified antibodies were stored as single-use aliquots at 4 °C until needed.
  • the particles were then washed by centrifugation (2000 rpm, 10 min) and re-dispersed with PBS.
  • 10 ⁇ of monoclonal anti-PSA developed in mouse (0.13 mg mL "1 ) was added to detect the PSA with a sandwich immunoassay format.
  • the particles were washed and re-dispersed in PBS and 20 [it of GOx-labeled anti-mouse IgG added for 2 h at room temperature. After washing once with MES buffer containing Tween-20 (0.05%) and once with MES buffer, the signal generation step was carried out as described above.
  • XEDS X-ray energy dispersive spectroscopy
  • Figure 6 shows the XEDS spectrum of free-standing silver nanoparticles obtained with 10 "14 g-mL ' 1 GOx.
  • Figures 7, 8 and 9 show XEDS spectra obtained from gold nanostars modified with 0, 10 " 20 and 10 "14 g-mL '1 GOx, respectively after the signal amplification step. Each experiment was repeated 10 times at random sites of the grid with identical results.
  • Figure 6 shows that the freestanding round nanoparticles are made of silver but not gold, which demonstrates that they are generated via nucleation in solution triggered by the biocatalytic activity of GOx.
  • the XEDS spectrum obtained with gold nanostars that were not modified with GOx after addition of glucose and silver does not show any signal for silver.
  • serum from female donors may contain PSA.
  • concentration of endogenous PSA in the serum used in Figure 2b can be estimated from the experiments in the absence of PSA performed in PBS and serum, as shown in Figure 10 and 1 1. It should be noted that this is an approximation because we are not taking into account the contribution of nonspecific interactions in this calculation. After calculating the difference in the LSPR absorbance position for both cases and interpolating this value in the calibration curve shown in Figure 2a, it was estimated that the serum from the female donor used here contains approximately 10 " g mL "1 PSA. The same serum from the same batch extracted from the same donor was used for all experiments.
  • gold nanostars were covalently modified with GOx (as described above). After adding glucose to trigger the enzymatic production of hydrogen peroxide, silver nitrate was added to initiate crystal growth.
  • the LSPR of gold nanostars modified with 10 "14 g mL "1 GOx undergoes a blue shift after silver reduction, as expected from the formation of a silver coating around gold nanosensors. 13 ' 17-18
  • the same experiment performed with gold nanostars modified with a non-catalytic globular protein (BSA) does not alter the optical properties of the nanosensors, which indicates that the presence of GOx is essential for the formation of a silver coating.
  • BSA non-catalytic globular protein
  • the magnitude of the signal registered by plasmonic nanosensors depends on the rate of crystallization, which favours either the growth of a silver coating on the existing nanocrystals or the nucleation of free-standing small particles.
  • the presence of silver nanostructures in the solutions containing GOx-modified nanostars was determined after the crystal growth step.
  • Figures 3a and 3b show representative TEM images obtained after silver reduction in the presence of 10 "20 g mL "1 and 10 " 14 g mL "1 GOx, respectively.
  • XEDS X-ray energy dispersive spectroscopy
  • the calibration curve for the detection of PSA in buffered solution shows the negative slope that is characteristic of inverse sensitivity in the concentration range between 10 "18 and 10 "13 g mL "1 (see also Figures 10 and 11 ).
  • the limit of detection defined here as the lowest concentration of analyte in the inverse sensitivity regime, was 10 "18 g mL "1 .
  • the signal generation time is longer (ca. 3 h), this result is one order of magnitude lower compared to recently proposed ultrasensitive digital ELISA assays. 7
  • the same experiments performed with a control protein (BSA) did not yield any significant signal, which proved that the effect of nonspecific interactions between the GOx-labeled antibodies and the nanosensors were minimal.
  • Nanoparticle-based bio-barcode assay redefines "undetectable” PSA and biochemical recurrence after radical prostatectomy. Proc. Natl. Acad. Sci. USA 106, 18437- 18442 (2009)
  • Adsorbate-Surface Bonding Simple Alkenes and Alkynes Adsorbed at Gold Electrodes. J. Phys. Chem. 89, 5046-5051 (1985)

Abstract

Cette invention concerne une méthode permettant de déterminer la présence d'un analyte dans un échantillon liquide, la méthode comprenant la mise en contact d'un premier fragment de liaison avec l'échantillon liquide, le premier fragment de liaison étant lié à un fragment signal qui génère un signal et se liant spécifiquement à l'analyte pour former un conjugué réactionnel. Un conjugué de détection est formé par liaison du conjugué réactionnel à un fragment générant un agent de réduction. Le conjugué de détection est mis en contact avec un substrat pour le fragment générant l'agent de réduction et des ions métalliques ; et le signal généré par le fragment signal est mesuré, toute variation du signal indiquant la présence d'un analyte.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104792766A (zh) * 2015-04-15 2015-07-22 江苏理工学院 表面增强拉曼散射基底及其制备方法
US9903868B2 (en) 2014-05-16 2018-02-27 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Method for the detection and quantitation of biomarkers
KR20190112783A (ko) * 2017-01-30 2019-10-07 아박시스, 인크. 용액-기반 플라스몬 특이적-결합 파트너 검정 및 금속성 나노구조체
US11614447B2 (en) 2015-08-04 2023-03-28 Zoetis Services Llc Signal amplification in solution-based plasmonic specific-binding partner assays

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0120694A2 (fr) 1983-03-25 1984-10-03 Celltech Limited Procédés pour la production des polypeptides ou protéines à chaînes multiples
EP0125023A1 (fr) 1983-04-08 1984-11-14 Genentech, Inc. Préparations d'immunoglobuline recombinante, méthodes pour leur préparation, séquences d'ADN, vecteurs d'expression et cellules d'hôtes recombinantes
EP0184187A2 (fr) 1984-12-04 1986-06-11 Teijin Limited Chaîne lourde d'immunoglobuline chimère souris-humaine et chimère de l'ADN codant celle-ci
EP0239400A2 (fr) 1986-03-27 1987-09-30 Medical Research Council Anticorps recombinants et leurs procédés de production
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
WO1994013804A1 (fr) 1992-12-04 1994-06-23 Medical Research Council Proteines de liaison multivalentes et multispecifiques, leur fabrication et leur utilisation
US9209965B2 (en) 2014-01-14 2015-12-08 Microsemi Semiconductor Ulc Network interface with clock recovery module on line card

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0120694A2 (fr) 1983-03-25 1984-10-03 Celltech Limited Procédés pour la production des polypeptides ou protéines à chaînes multiples
EP0125023A1 (fr) 1983-04-08 1984-11-14 Genentech, Inc. Préparations d'immunoglobuline recombinante, méthodes pour leur préparation, séquences d'ADN, vecteurs d'expression et cellules d'hôtes recombinantes
EP0184187A2 (fr) 1984-12-04 1986-06-11 Teijin Limited Chaîne lourde d'immunoglobuline chimère souris-humaine et chimère de l'ADN codant celle-ci
EP0239400A2 (fr) 1986-03-27 1987-09-30 Medical Research Council Anticorps recombinants et leurs procédés de production
GB2188638A (en) 1986-03-27 1987-10-07 Gregory Paul Winter Chimeric antibodies
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
WO1994013804A1 (fr) 1992-12-04 1994-06-23 Medical Research Council Proteines de liaison multivalentes et multispecifiques, leur fabrication et leur utilisation
US9209965B2 (en) 2014-01-14 2015-12-08 Microsemi Semiconductor Ulc Network interface with clock recovery module on line card

Non-Patent Citations (38)

* Cited by examiner, † Cited by third party
Title
AILI, D.; STEVENS, M. M.: "Bioresponsive peptide-inorganic hybrid nanomaterials", CHEM. SOC. REV., vol. 39, 2010, pages 3358 - 3370
BARBOSA, S. ET AL.: "Tuning Size and Sensing Properties in Colloidal Gold Nanostars", LANGMUIR, vol. 26, 2010, pages 14943 - 14950
BATT, C. A.: "Food Pathogen Detection", SCIENCE, vol. 316, 2007, pages 1579 - 1580
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426
CARDINAL, M. F.; RODRIGUEZ-GONZALEZ, B.; ALVAREZ-PUEBLA, R. A.; PEREZ-JUSTE, J.; LIZ-MARZAN, L. M.: "Modulation of Localized Surface Plasmon and SERS response in Gold Dumbbells through Silver Coating", J. PHYS. CHEM. C., vol. 114, 2010, pages 10417 - 10423
CHANG, Y.-F.; HUNG, S.-H.; LEE, Y.-J.; CHEN, R.-C.; SU, L.-C.; LAI, C.-S.; CHOU, C., ANAL. CHEM., vol. 83, 2011, pages 5324 - 5328
CHEN ET AL: "Successively amplified electrochemical immunoassay based on biocatalytic deposition of silver nanoparticles and silver enhancement", BIOSENSORS AND BIOELECTRONICS, ELSEVIER BV, NL, vol. 23, no. 4, 17 October 2007 (2007-10-17), pages 485 - 491, XP022302118, ISSN: 0956-5663, DOI: 10.1016/J.BIOS.2007.06.005 *
CHEN SI ET AL: "Plasmon-enhanced colorimetric ELISA with single molecule sensitivity.", NANO LETTERS 13 APR 2011 LNKD- PUBMED:21428275, vol. 11, no. 4, 13 April 2011 (2011-04-13), pages 1826 - 1830, XP009164080, ISSN: 1530-6992 *
DE LA RICA, R.; BALDI, A.; FERNANDEZ-SANCHEZ, C.; MATSUI, A.: "Selective Detection of Live Pathogens via Surface-Confined Electric Field Perturbation on Interdigitated Silicon Transducers", ANAL. CHEM., vol. 81, 2009, pages 3830 - 3835
DE LA RICA, R.; BALDI, A.; FERNANDEZ-SANCHEZ, C.; MATSUI, H.: "Single-Cell Pathogen Detection with a Reverse-Phase Immunoassay on Impedimetric Transducers", ANAL. CHEM., vol. 81, 2009, pages 7732 - 7736
DE LA RICA, R.; MENDOZA, E; MATSUI H.: "Bioinspired Target-Specific Crystallization on Peptide Nanotubes for Ultrasensitive Pb Ion Detection", SMALL, vol. 6, 2010, pages 1753 - 1756
ENDO T ET AL: "Localized surface plasmon resonance based optical biosensor using surface modified nanoparticle layer for label-free monitoring of antigen-antibody reaction", SCIENCE AND TECHNOLOGY OF ADVANCED MATERIALS, ELSEVIER, OXFORD, GB, vol. 6, no. 5, 1 July 2005 (2005-07-01), pages 491 - 500, XP027624487, ISSN: 1468-6996, [retrieved on 20050701] *
FAN, R. ET AL.: "Integrated barcode chips for rapid, multiplexed analysis of proteins in microliter quantities of blood", NAT. BIOTECHNOL., vol. 26, 2008, pages 1373 - 1378
FERBER, D.: "Overhaul of CDC Panel Revives Lead Safety Debate", SCIENCE, vol. 298, 2002, pages 732 - 732
G.T. HERMANSON: "Bioconjugate Techniques", 1996, ACADEMIC PRESS, INC., pages: 57 - 60
GILJOHANN D. A.; MIRKIN C. A.: "Drivers of biodiagnostic development", NATURE, vol. 462, 2009, pages 461 - 464
HAES A J ET AL: "DETECTION OF A BIOMARKER FOR ALZHEIMER'S DISEASE FROM SYNTHETIC AND CLINICAL SAMPLES USING A NANOSCALE OPTICAL BIOSENSOR", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, ACS PUBLICATIONS, US, vol. 127, no. 7, 23 February 2005 (2005-02-23), pages 2264 - 2271, XP002568084, ISSN: 0002-7863, [retrieved on 20050127], DOI: 10.1021/JA044087Q *
HOLLINGER; WINTER, CURRENT OPINION BIOTECHNOL., vol. 4, 1993, pages 446 - 449
HUSTON ET AL., PNAS USA, vol. 85, 1988, pages 5879 - 5883
J. T. SEO ET AL.: "Optical nonlinearities of Au Nanoparticles and Au/Ag coreshells", OPT. LETT., vol. 34, 2009, pages 307 - 309
JANA, N. R.; GEARHEART, L.; MURPHY, C. J.: "Evidence for Seed-Mediated Nucleation in the Chemical Reduction of Gold Salts to Gold Nanoparticles", CHEM. MATER., vol. 13, 2001, pages 2313 - 2322
KOBAYASHI, K.; OKAMOTO; MORITA, N.; KIYOTANI, T.; TAMURA, O.: "Synthesis of the proposed structure of phaeosphaeride A", ORG. BIOMOL. CHEM., vol. 9, 2011, pages 5825 - 5832
KUMAR, P. S.; PASTORIZA-SANTOS, I.; RODRIGUEZ-GONZALEZ, B.; GARCIA DE ABAJO, F. J.; LIZ-MARZAN, L. M.: "High-yield synthesis and optical response of gold nanostars", NANOTECHNOLOGY, vol. 19, 2007, pages 015606
LAROMAINE, A.; KOH, L. L.; MURUGESAN, M.; ULIJN, R. V.; STEVENS, M. M.: "Protease-triggered dispersion of nanoparticle assemblies", J. AM. CHEM. SOC., vol. 129, 2007, pages 4156 - 4157
LI, D.; WIECKOWSKA, A.; WILLNER, .: "Optical analysis of Hg(2+) ions by oligonucleotide-gold- nanoparticle hybrids and DNA-based machines", ANGEW. CHEM. INT. ED., vol. 47, 2008, pages 3927 - 3931
LIU, M.; GUYOT-SIONNEST, P.: "Mechanism of Silver(I)-assisted Growth of Gold Nanorods and Bipyramids", J. PHYS. CHEM. B, vol. 109, 2005, pages 22192 - 2220
LIZ-MARZAN, L.; M. TAILORING: "Surface Plasmons through the Morphology and Assembly of Metal Nanoparticles", LANGMUIR, vol. 22, 2006, pages 32 - 41
MIRANDA, O. R. ET AL.: "Enzyme-Amplified Array Sensing of Proteins in Solution and in Biofluids", J. AM. CHEM. SOC., vol. 132, 2010, pages 5285 - 5289
MOSKOVITS, M.; DILELLA, D. P.; MAYNARD, K. J.: "Surface Raman-spectroscopy of a number of cyclic aromatic-molecules adsorbed on silver - Selection-rules and molecular-reorientation", LANGMUIR, vol. 4, 1988, pages 67 - 76
P. HOLLINGER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 6444 - 6448
PATTERSON, M. L.; WEAVER, M. J.: "Surface-Enhanced Raman Spectroscopy as a Probe of Adsorbate-Surface Bonding: Simple Alkenes and Alkynes Adsorbed at Gold Electrodes", J. PHYS. CHEM., vol. 89, 1985, pages 5046 - 5051
RISSIN, D. M. ET AL.: "Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations", NAT. BIOTECHNOL., vol. 28, 2010, pages 596 - 599
RODRIGUEZ-LORENZO, L.; ALVAREZ-PUEBLA, R. A.; DE ABAJO, F. J. G.; LIZ-MARZAN, L. M.: "Surface Enhanced Raman Scattering Using Star-Shaped Gold Colloidal Nanoparticles", J. PHYS. CHEM. C, vol. 114, 2010, pages 7336 - 7340
THAXTON, C. S. ET AL.: "Nanoparticle-based bio-barcode assay redefines ''undetectable'' PSA and biochemical recurrence after radical prostatectomy", PROC. NATL. ACAD. SCI. USA, vol. 106, 2009, pages 18437 - 18442
TRAUNECKER ET AL., EMBO JOURNAL, vol. 10, 1991, pages 3655 - 3659
TUMA, R.: "Raman spectroscopy of proteins: from peptides to large assemblies", JOURNAL OF RAMAN SPECTROSCOPY, vol. 36, 2005, pages 307 - 319
WARD, E.S. ET AL., NATURE, vol. 341, 1989, pages 544 - 546
XU X ET AL: "Application and Research Development of Surface Plasmon Resonance-based Immunosensors for Protein Detection", CHINESE JOURNAL OF ANALYTICAL CHEMISTRY,, vol. 38, no. 7, 1 July 2010 (2010-07-01), pages 1052 - 1059, XP027248242, ISSN: 1872-2040, [retrieved on 20100701] *

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