WO2015055708A1 - Dosage biologique qualitatif sensible ay moyen d'oxyde de graphène comme agent révélateur d'analyte - Google Patents

Dosage biologique qualitatif sensible ay moyen d'oxyde de graphène comme agent révélateur d'analyte Download PDF

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WO2015055708A1
WO2015055708A1 PCT/EP2014/072100 EP2014072100W WO2015055708A1 WO 2015055708 A1 WO2015055708 A1 WO 2015055708A1 EP 2014072100 W EP2014072100 W EP 2014072100W WO 2015055708 A1 WO2015055708 A1 WO 2015055708A1
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analyte
binding molecule
comprised
mixture
qds
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PCT/EP2014/072100
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Arben MERKOÇI HYKA
Eden MORALES NARVÁEZ
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Fundació Institut Català De Nanociència I Nanotecnologia
Institució Catalana De Recerca I Estudis Avançats
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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • 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
    • G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/245—Escherichia (G)

Definitions

  • the present invention belongs to the field of analytical chemistry.
  • the invention is particularly referred to a biosensing method for detecting analytes, particularly pathogens, by taking advantage of the optical qualities of quantum dots and graphene oxide.
  • the culturing and colony counting technique is the oldest bacterial detection method. Different selective media are used to detect particular bacterial species. The media often contain inhibitors to stop or delay growth of non- target strains or particular substrates that only the targeted bacteria can degrade. When the colonies have grown, detection is carried out using optical methods, mainly by ocular inspection. Despite remaining the standard method for bacterial detection, culturing and colony counting technique is excessively time consuming (at least 2-9 days are needed for bacterial growth) and usually requires specialised staff and instrumentation. Further, the technique requires that serial dilutions are made when the initial bacterial concentration of the sample is very high, while it is not appropriate to detect very low bacterial numbers.
  • PCR is a nucleic acid amplification technique which is also widely used for bacterial detection. It is based on the isolation, amplification and detection of a short DNA sequence that should be specific of the targeted bacteria. PCR- based techniques have become very popular for their versatility, but are also time-consuming. It takes from 5 to 24 hours to produce a detection result, and this does not include any previous enrichment steps that might be necessary. Further, these methods are not devoid of manipulation and need of specialised technical staff.
  • Immunology based methods provide detection of a wide range of bacterial targets.
  • the most well stablished immunological based technique for pathogen detection is enzyme-linked immunosorbent assay (ELISA).
  • ELISAs combine the specificity of antibodies and the sensitivity of simple enzyme assays by using antibodies or antigens coupled to easily assayed enzymes.
  • Traditional ELISAs are less time consuming that the above techniques - results being obtained in the range from 20 min to 6 hours- but, nevertheless, they require a lot of manipulation.
  • Newer modalities, such as lateral flow immunochromatographic methods may be quicker and also require little manipulation. However, they lack for sensitivity and often provide false positive or negative results.
  • an engineered gold nanoparticle-labeled antibody probe was attached to the captured target cell whose complexes enable gold nanoparticles to be close to the GO surface, thereby resulting in the quenching of GO fluorescence signal to identify the pathogen.
  • An increasing number of NPs leads to drastic fluorescence reduction of GO, allowing rotavirus detection with a limit of detection of 10 4 colony forming units/mL (CFU/mL).
  • CFU/mL colony forming units/mL
  • the inventors have developed a high performance bioassay for detecting the presence of pathogenic microorganisms with surprisingly high sensitivity, while being quick, easy to use, down-scalable and highly specific.
  • the assay is advantageous for detecting any type of analyte as long as its size is 20 nm or greater and takes advantage of the FRET process that occurs between fluorescent QDs and GO, when the GO is used as pathogen revealing agent.
  • a first aspect of the invention provides a method for detecting an analyte of size comprised from 20 to 10000 nm in a mixture, the method comprising: (i) contacting the analyte with a quantum dot that is conjugated to at least one binding molecule, wherein the binding molecule has affinity for the analyte which means that the analyte is selectively captured by the binding molecule, (ii) determining the fluorescence intensity of the mixture of step (i) upon exposing the mixture to an electromagnetic radiation capable of exciting the quantum dot, (iii) adding graphene oxide to the mixture of step (ii), and (iv) determining the fluorescence intensity of the mixture of step (iii) upon exposing the mixture to the same electromagnetic radiation used in step (ii), wherein a reduction in fluorescence intensity of the mixture of step (iii) compared with that of step (i) is indicative of the absence of the analyte.
  • the method of the invention may detect a pathogenic bacteria which is present in a sample at a concentration as small as 5 CFU/mL. On top of this surprisingly high sensitivity, the method may provide results in a very short time. Due to its convenient configuration, the method is easy to use and versatile. It can be adapted to many types of formats, including solid supports such as microarray plates, microtiter plates or immunochromatographic strips. The method can also proceed in solution. This versatility includes multiplex detection using standard microarray equipment. It is also possible to down- scale the assay to a small device for point of use applications which would not have need of complex laboratory equipment or specially trained staff.
  • FIG. 1 represents a particular embodiment of the invention where the binding molecule is an antibody (Ab) specific for E. coli .
  • Ab an antibody
  • the (bound) probes barely interact with the GO; consequently, the GO only minimally quenches their fluorescence (ON state, 1 ), whereas in the absence of the pathogen (A), the probes are quenched by electrostatic n-n stacking interaction between the probes and the GO (OFF state, 0).
  • the selectively captured analyte does not allow for efficient FRET energy transfer between the QDs and GO, so that minimal variations on the fluorescent intensities of the QDs indicate that the analyte is present in the sample while a sharp reduction of
  • the blank signal is the fluorescence intensity of the probes when incubated in the absence of an analyte.
  • the method of the invention yielded a limit of detection (LOD) of 5 CFU/mL (see examples below and FIGs. 4 and 5). This means that when more than 5 CFU/mL of E. coli cells were present in the sample, the fluorescence intensity of the QDs suffered a minimal, nonsignificant variation (ON state). On the contrary, when less han 5 CFU/mL of E. coli cells were present in the sample, a sharp decrease of the
  • the assay has been adapted to a solid-support microarray format and the detection takes place in the solid state using standard microarray instrumentation.
  • the QD probe i.e. using a different binding molecule conjugated to the QD
  • multiplex detection of hundred different analytes is achieved.
  • multiplex detection of the same analyte in tens of different samples is achieved.
  • the qualitative bioassay of the invention has a pathogenic effect.
  • interaction of the GO revealing agent with the bacterial cell may kill the bacteria by compromising the integrity of the bacterial membrane, thus comprising a biosensing and microbicidal GO-based system that may both detect bacteria and kill them.
  • the inventors have also found that detection of analytes may succeed with the same advantages mentioned above by contacting said analyte to a spacer particle, wherein said spacer particle has a size comprised from 20 to 10000 nm and is optically inactive.
  • a spacer particle advantageously enables detection of analytes of size smaller than 20 nm, such as proteins, nucleic acids and viruses.
  • analyte of size comprised from 1 to 10000 nm in a mixture
  • the method comprising: (i) contacting the analyte with (a) a quantum dot that is attached to a solid support and conjugated to at least one first binding molecule, wherein the first binding molecule has affinity for the analyte, and (b) optionally a spacer particle that is conjugated to at least one second binding molecule, wherein the second binding molecule has affinity for the analyte and may be identical or different to the first binding molecule, and wherein the spacer particle has a size comprised from 20 nm to 10000 nm and is optically inactive, wherein when step (b) is performed steps (a) and (b) are performed in any order; (ii) determining the fluorescence emission intensity of the mixture of step (i) upon exposing the mixture to an electromagnetic radiation capable of exciting the quantum dot; (iii) adding graphene oxide to
  • Figure 6 illustrates the working of the method of the invention when a spacer particle is used.
  • the figure represents a particular embodiment where the binding molecules are antibodies and the spacer particle is a silica bead.
  • Antibody coated QDs (E) are used to capture the analyte (F).
  • FRET analyte
  • the probes are quenched by FRET between QDs and GO (A). This state is known as OFF state (0).
  • detection antibodies conjugated to silica beads are used to avoid FRET. This state is termed ON state (1 ).
  • the selectively captured analyte and spacer particle do not allow for efficient FRET energy transfer between the QDs and GO, so that minimal variations on the fluorescent intensities of the QDs indicate that the analyte is present in the sample. In contrast, a sharp reduction of differences on fluorescence intensity of the QDs is indicative of the absence of the analyte.
  • the invention provides a kit for carrying out the method of the invention, which comprises adequate means and instructions for carrying out the method, wherein the means comprise at least QD-binding molecule probes and graphene oxide, wherein said graphene oxide: (a) contains an atomic carbon to oxigen ratio (C/O) comprised from 1 to 5, preferably from 1 to 2, (b) contains a lateral size comprised from 5 to 1000 nm, and (c) contains from 1 to 20, preferably 1 to 10, layers.
  • the kit may also include a solid support, spacer particles and other reagents and/or buffers.
  • FIG. 1 Operational concept of the qualitative bioassay of the invention (illustration not to scale).
  • C graphene oxide
  • D E. coli
  • E antibody-QD conjugate
  • G capture spot
  • F chip on glass slide
  • A in the absence of analyte
  • 0 OFF state
  • B in the presence of analyte (E. coli); 1 : ON state.
  • FIG. 2 Scanning electron microscopy (SEM) images of the explored system.
  • a Ab-QD complexes inside a microspot.
  • b Ab-QD/GO complexes inside a microspot.
  • c Ab-QD/E. coli (membrane-bound)/GO complexes inside a microspot. Aminosilane silicon slides were used as substrate.
  • FIG. 3 a. Spot profile of the system's response. The dotted lines denote the original profile of the explored spots. The solid lines indicate the final profile of the observed spots at several concentrations of the target pathogen, E. coli ([Ec] in CFU/mL) after GO addition, b. In the absence of E.
  • E. coli [Ec] in CFU/mL
  • the spots which comprise Ab-QD probes, are quenched by adding GO (blank signal).
  • the control signal represents the maximum signal that the system can display: this corresponds to the probes being incubated with a blank throughout the assay (i.e. without adding GO).
  • FIG. 4 Performance of the Ab-QD pathogen-detection system in the transition zone at E. coli concentrations [Ec] of 0 to 100 CFU/mL, in PBS (b') and in tap water (b").
  • FIG. 5 Response of the qualitative bioassay of the invention (using GO as the pathogen-revealing agent, ⁇ ) as compared to an immunosandwich configuration (using Cy3- labelled antibody as the pathogen-revealing agent, x). The error bars were obtained from parallel assays of 10 microspots in each configuration. The dotted line corresponds to the threshold of the LOD. l/l 0 : final intensity/original intensity. N: normalised intensity.
  • FIG. 6 Operational concept of the qualitative bioassay of the invention when using a spacer particle (illustration not to scale).
  • C graphene oxide
  • D E. coli
  • E antibody-QD conjugate
  • G capture spot
  • F chip on glass slide
  • A in the absence of analyte
  • 0 OFF state
  • B in the presence of analyte (E. coli); 1 : ON state.
  • QDs quantum dots
  • a QD is understood in the state of the art as photoluminescent semiconductor nanocrystals whose characteristics are closely related to the size and shape of the individual crystal.
  • a QD is capable of emitting electromagnetic radiation or light upon excitation (i.e., the QD is luminescent).
  • the smaller the size of the crystal the larger the band gap, the greater the difference in energy between the highest valence band and the lowest the conduction band becomes, therefore more energy is needed to excite the dot, and concurrently, more energy is released when the crystal returns to its resting state.
  • fluorescent dye for example, in fluorescent dye
  • QDs have broad excitation spectra with narrow emission bandwidths that span the visible spectrum, allowing simultaneous excitation of several particle sizes at a single wavelength. QDs also have exceptional photochemical stability. Altogether, QDs are an advantageous alternative to conventional organic dyes for bioanalytical applications.
  • a QD is comprised of a first semiconductor material that sometimes can be coated by a second semiconductor material.
  • the coating material will preferably have a bandgap energy that is larger than the bandgap energy of the first material and may be chosen to have an atomic spacing close to that of the first material.
  • QD size is usually comprised from about 1 nm to about 50 nm in diameter, sometimes from 2 nm to 20 nm, sometimes from 2 nm to 10 nm.
  • QDs may be prepared by methods well established in the state of the art.
  • Graphene oxide is a sheet-like compound of carbon, oxygen, and hydrogen in variable ratios, obtained by treating graphite with strong oxidizers.
  • Graphene oxide is available at variable C/O ratios (ratio expressed as carbon atoms related to oxigen atoms), usually from 1 to 5, and may be found in the form of single or multiple sheets (multiple in this case usually meaning from 2 to 20 layers).
  • oxygenated functional groups such as carboxyl (at the lattice edges), ester, hydroxyl or epoxide (on the basal plane of the lattice)
  • GO constitutes an oxygenated lattice of
  • Fluorescence refers to the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation. Many QDs are fluorescent.
  • Form fluorescence resonance energy transfer or “FRET” refers to a process based on two photoluminescent substances where initially one of the substances (the donor) can transfer energy to the second one (the acceptor). Generally, the efficiency of this energy transfer process is inversely
  • Electromagnetic radiation is a form of energy emitted and absorbed by charged particles which exhibits wave-like behavior as it travels through space. Electromagnetic radiation of any wavelenght is contemplated by the present invention but, particularly, the invention is referred to visible light and ultraviolet radiation.
  • binding molecule is meant a molecule or molecule segment capable of binding specifically to the target. Non-limitative binding molecules in the sense of the present invention are antibodies, antibody fragments, aptamers, molecular beacons, etc, all of which are binding molecules well known in the state of the art as being useful for bioanalytical applications.
  • antibody is meant a whole antibody, including without limitation a chimeric, recombinant, transgenic, humanised, grafted and single chain antibody, and the like, as well as any fusion protein, conjugates, fragments, or derivates thereof that contain one or more domains that selectively bind the target protein or peptide.
  • An antibody fragment means an Fv, a disulfide linked Fv, scFv, Fab, Fab', or F(ab')2 fragment, which are well known in the art.
  • Fab, Fv, ScFv and dAb antibody fragments can be expressed in and secreted from E. coli , thus allowing the facile production of large amounts of the fragments.
  • the term "spacer particle” refers to particle of size comprised from 20 nm to 10000 nm that conects the analyte and the GO revealing agent, providing a larger separation distance between the QD capturing the analyte and the GO.
  • the spacer particle is particularly important for detecting analytes of size smaller than 20 nm with high sensitivity. For such small analytes, the presence of a spacer particle determines that the QDs and the GO are separated at a distance that prevents FRET between them.
  • the spacer particles are optically inactive (transparent, so that they do not interfere in the FRET between QDs and GO) and susceptible to biofunctionalization (so that they may be conjugated with binding molecules).
  • Spacer particles for use in the present invention are, for example, silica beads, magnetic silica beads, nanocellulose particles, indium tin oxide particles, ZnS particles, transparent exopolymer particles, titania (titanium oxide) particles, Zinc oxide particles and ceria (cerium oxide) particles.
  • the inventors have developed a sensitive and versatile qualitative bioassay .
  • the fluorescence intensity of the QD probes is quenched by GO in the absence of the target analyte (OFF state).
  • GO Due to its oxygen-containing functional groups, GO can interact non- covalently with the diol, amino and phenyl groups present in the QD-binding molecule probe, for example with the amino groups of an antibody. This interaction brings GO in the proximity of the QDs, thus allowing for FRET energy transfer and efficient quenching of QD emission.
  • the target analyte for instance, a bacteria
  • the greater affinity of the binding- molecule for its target (bacteria) avoids efficient FRET between the QDs and GO, and consequently, QD emission is not quenched (ON state).
  • GO Owing to its high density of functional groups, GO can interact with bacteria, resulting in bacterial cell deposition. However, the size of the bacterial analyte precludes quenching of QD emission by the deposited GO.
  • any type of analyte may be detected with the bioassay of the invention, regardless of its chemical composition, as long as its size is comprised from 1 to around 10000 nm.
  • the size is comprised from 20 to 10000 nm, from 30 to 10000 nm or from 50 to 10000 nm.
  • the size of the analyte is comprised from 1 to 19 nm.
  • the method of the invention comprises use of a spacer particle.
  • Non-limiting target analytes for the qualitative bioassay of the invention are bacteria, virus, eukaryotic cells, proteins, large organic molecules and heavy metal-containing complexes.
  • the qualitative bioassay of the invention may be of particular interest in the food, environmental and/or medical fields for detecting pathogens (E. coli , Salmonella, virus, etc), cancer cells, disease-relevant proteins (such as beta amyloid, prostate specific antigen, etc), environmentally or food toxic proteins, biomarker-containing complexes, and environmentally-toxic organic molecules
  • pathogens E. coli , Salmonella, virus, etc
  • cancer cells cancer cells
  • disease-relevant proteins such as beta amyloid, prostate specific antigen, etc
  • environmentally or food toxic proteins such as biomarker-containing complexes
  • biomarker-containing complexes such as biomarker-containing complexes, and environmentally-toxic organic compounds
  • target analyte is a pathogenic bacteria.
  • target analyte is E. coli .
  • the QD probes of the qualitative bioassay of the invention fluoresce when excited by an appropriate electromagnetic radiation. After incubating the sample and adding GO, the intensity of the emmited light greatly differs in the absence or the presence of the analyte, and the fluorescent intensity variation allows for sensitive analyte detection.
  • the QDs for use in the present invention usually have emission wavelengths comprised from 400 to 750 nm.
  • the QDs have emission wavelenghts comprised from 500 to 720 nm, or from 600 to 700.
  • the emission wavelenght of the QDs is 670 nm.
  • the QDs are selected from the group
  • CdSe consisting of carbon, graphene, CdSe, CdS, or may have a core/shell structure, for example, CdSe coated with ZnS (CdSe/ZnS), CdSe coated with
  • CdS CdSe/CdS
  • CdS coated with ZnS CdS/ZnS
  • the coating (shell) of the QDs may be single (only one shell) or multishell.
  • the QDs In order to emit flourescence, the QDs must be excited by being exposed to an electromagnetic radiation of shorter wavelenght than that of the QDs' emission.
  • the wavelenght of the exciting electromagnetic radiation is from 40 to 25 nm, for example 28, 29, 30, 31 , 32, 33, 34 or 35 nm, shorter than the emission wavelenght of the QDs.
  • the wavelenght of the exciting electromagnetic radiation is from 40 to 25 nm, for example 28, 29, 30, 31 , 32, 33, 34 or 35 nm, shorter than the emission wavelenght of the QDs.
  • electromagnetic radiation capable of exciting the quantum dot has a wavelength comprised from 360 to 725 nm. In other embodiments, the electromagnetic radiation capable of exciting the quantum dot has a wavelength comprised from 450 to 700 nm or from 550 to 680 nm. In a particular embodiment, the electromagnetic radiation capable of exciting the quantum dot has a wavelength of 633 nm. Any source may be employed for producing the excitation electromagnetic radiation, for example, a laser, light- emitting diode (LED) lamp, ultra-violet (UV) lamp.
  • LED light- emitting diode
  • UV ultra-violet
  • the QDs When excited, the QDs thus emmit a read-out signal, which will vary depending on the presence or the absence of the analyte as detailed above.
  • the read-out signal is recorded by a fluorometer, imaging system, or any other device used to measure parameters of fluorescence, by fixing the emmision filter at the predetermined emmision of the QDs.
  • the fluorometer thus records the emmision intensity of the QDs to yield a result (presence or absence of the analyte).
  • Any fluorometer or imaging system may be appropriate for use in the qualitative bioassay of the invention. The type used will depend on the configuration of the assay (in solution, in microtiter plates, in microarray plates, in paper, etc). In a particular embodiment, the
  • fluorometer is a microarray scanner.
  • the QDs are conjugated to binding molecules which have high affinity for the target analyte, such that incubating of an analyte-containing sample results in the analyte being selectively captured by the binding molecule.
  • conjugation it is generally meant the joining together of two compounds resulting in the formation of another compound, in this case, the joining together of the QD and the binding molecule.
  • the binding molecule is an antibody, an antibody fragment, a molecular beacon or an aptamer, all of which may be obtained by standard techniques.
  • QDs need to be functionalised.
  • the QDs need to be water soluble for biological applications.
  • they need to be biofunctionalized in order to meet four key requirements: (1 ) increased stability in water for long period, (2) presence of sterically accessible functional groups for bioconjugation, (3) biocompatibility and non-immunogenicity in living systems, and (4) lack of interference with the QD native properties.
  • Solubilisation and biofunctionalisation of QDs is achieved by methods well known in the art. These methods are comprehensively summarised, for example in the following reviews: Medintz I, et al, "Quantum dot bioconjugates for imaging, labelling and sensing", Nature Materials, 2005, vol. 4, p. 435, and Mazumder S, et al, "Biofunctionalized Quantum Dots in Biology and
  • the QD-binding molecule probes are obtained by the streptavidin-biotin approach, for which steptavidin-covered QDs are contacted with biotinylated binding molecules, for example, biotinylated antibodies (see examples below).
  • the QDs may be capped by a silane shell and functionalised with, for instance, a compound providing amine, sulfhydryl or carboxy functionality which can react with a peptidic or DNA binding molecule.
  • the method of the invention requires use of a spacer particle as defined above.
  • the spacer particles are also conjugated to binding molecules which have high affinity for the target analyte.
  • the binding molecule conjugated to the spacer particle may be the same or different to the binding molecule that is conjugated to the QD.
  • the binding molecule is an antibody, an antibody fragment, a molecular beacon or an aptamer, all of which may be obtained by standard techniques.
  • spacer particles need to be surface-functional ised.
  • Biofunctionalisation of the surface of particles, particularly nanoparticles such as silica beads or nanocellulose beads, is achieved by methods well known in the art (Froimowicz P., et al. "Surface-Functional ized Particles: From their Design and Synthesis to
  • the spacer particle may be functionalised with a compound providing amine, sulfhydryl or carboxy functionality which can react with a peptidic or DNA binding molecule.
  • Surface-functionalised particles can be readily obtained from commercial sources.
  • the spacer particle when used, may be contacted with the analyte before or after contacting the analyte with the QDs, but always before addition of the GO revealing agent.
  • the analyte is first incubated with the binding molecule-decorated QDs and then the binding molecule-decorated spacer particles are added to the previously formed QD-analyte complex.
  • the analyte is first incubated with the binding molecule- decorated spacer particle and then formed spacer particle-analyte complex is incubated with the binding molecule-decorated QD. Examples of the proceedure when using a spacer particle in the method of the invention are described in the examples below.
  • the QD-binding molecule probe is attached to a solid support.
  • the support may be, among other, glass, plastic, cellulose, nitrocellulose or paper. Attachment of the probe to the solid support may succeed by methods well known in the art (Angenendt P, Drug Discovery Today, 2005, vol. 10(7), p. 503).
  • the QD-binding molecule probe may be attached to two-dimensional plain glass slides, which are activated with a variety of coupling chemistries such as aldehyde, epoxy or carboxylic esters. Slides with these surfaces bind biomolecules (for example peptides or nucleic acids) either by electrostatic interactions or through the formation of covalent bonds.
  • the QD-binding molecule probe can be attached to three-dimensional gel or membrane-coated surfaces, such as polyacrylamide, agarose and nitrocellulose. These surfaces bind
  • the QD-binding molecule probe can be attached to surface coatings, such as dendrimer or avidin slides, which mix both concepts mentioned above.
  • the solid support is amino, epoxy or carboxi-functionalised.
  • the support comprises aminosilane glass surface.
  • the solid support has microarray format, meaning that QD probes are deposited or immobilised onto the solid support following a microarray design, i.e., the probes are densely spotted onto the support on a few square microns such that large number of samples may be analysed simultaneously.
  • immobilisation strategies known in the art may be employed to this end, including, among others, immobilisation of antibodies by DNA-directed immobilisation (DDI), direct spotting, and streptavidin-biotin attachment (Wacker, R. et al. "Performance of antibody microarrays fabricated by either DNA-directed immobilization, direct spotting, or streptavidinbiotin attachment: a comparative study". Anal. Biochem. 2004, vol.
  • microarray format comprises a glass surface.
  • the solid support has lateral flow format.
  • Lateral flow tests also known as lateral flow immunochromatographic assays, are simple devices intended to detect the presence (or absence) of a target analyte in a sample (matrix) without the need for specialized and costly equipment, though many lab based applications exist that are supported by reading equipment. These lateral flow tests are well known in the art and appropriate for point of use applications. Often they comprise a series of capillary beds, each of which has the capacity to transport fluid. The sample thus flows through the different capillary beds contacting the reagents so that a detection result is finally observed.
  • the solid support with lateral flow format comprises a nitrocellulose membrane to which the QD-binding molecule label is attached.
  • the solid support has microtiter plate format, such as those used for ELISA assays.
  • the GO is added at the final stage of the assay as revealing agent.
  • addition of GO allows for differentiation between the presence or absence of the analyte in the sample.
  • the GO contains an atom carbon to oxigen ratio (C/O) comprised from around 1 to around 5.
  • C/O ratio is comprised from 1 to 2, or from 1 to 1 .5, or from 1 to 1 .25.
  • the C/O ratio is 1 .
  • the GO in the sense of the present invention may comprise from 1 to 20 layers.
  • the GO comprises from 1 to 15, or from 1 to 10 or from 2 to 10 or from 1 to 5 or from 2 to 5 layers.
  • the lateral length of GO sheets may be comprised from 5 to 1000 nm. In one embodiment, the lateral length of GO sheets is comprised from 100 to 1000 nm. In one embodiment, the lateral length of GO sheets is comprised from 5 to 500 nm. In one
  • the lateral length of GO sheets is comprised from 50 to 500 nm. In one embodiment, the lateral length of GO sheets is comprised from 250 to 1000 nm. In another embodiment, the lateral length of GO sheets is
  • GO is added to the assay (specifically, step (iii) of the method of the first aspect of the invention as defined above) at a final concentration comprised between 0.01 to 400 pg/nnL.
  • GO is added at a final concentration comprised from 0.1 to 400 g/mL, or from 1 to 400 pg/nriL or from 1 to 250 pg/mL, or from 5 to 200 g/mL, or from 10 to 175 pg/mL, or from 20 to 150 pg/mL, or from 25 to 125 g/mL, or from 50 to 100 pg/mL, or rom 60 to 80 pg/nnL. In another particular embodiment GO is added at a final concentration of around 70 pg/nnL.
  • the GO added in step (iii) of the method defined by the first aspect of the invention contains an atomic carbon to oxigen ratio (C/O) comprised from 1 to 2, (b) contains a lateral size comprised from 5 to 1000 nm, (c) contains from 1 to 10 layers, and (d) is added at a final concentration comprised from 0.1 to 400 g /ml_.
  • C/O atomic carbon to oxigen ratio
  • GO in step (iii) contains an atomic carbon to oxigen ratio (C/O) comprised from around 1 .5 to around 2, (b) contains a lateral size comprised from 5 to 750 nm, (c) contains from 1 to 5 layers, and (d) is added at a final concentration comprised from 0.01 to 100 g /ml_.
  • C/O atomic carbon to oxigen ratio
  • GO in step (iii) (a) contains an atomic carbon to oxigen ratio (C/O) comprised from around 1 to around 1 .5, (b) contains a lateral size comprised from 5 to 750 nm, (c) contains from 1 to 15 layers, and (d) is added at a final concentration comprised from 0.1 to 200 g /ml_.
  • C/O atomic carbon to oxigen ratio
  • GO in step (iii) (a) contains an atomic carbon to oxigen ratio (C/O) comprised from 1 to 1 .25, (b) contains a lateral size comprised from 5 to 700 nm, (c) contains from 1 to 10 layers, and (d) is added at a final concentration comprised from 1 to 400 ig /mL.
  • the analyte containing sample must be placed in contact (or incubated) with the QD probes enough time for selective binding to take place between the sample and the binding molecule.
  • the incubation time depends on the analyte and the binding molecule. In one particular embodiment, incubating time of the sample with the probe is from 5 min to 180 min. In other words, incubating time of the sample with the probe is from 5 min to 180 min. In other words
  • the incubating time is from 15 to 180 min, or from 30 to 160 min, or from 45 to 120 min, or from 60 to 120 min. Further, as described above, detection of the analyte succeeds after addition of the GO (which acts as analyte-revealing agent). In some embodiments the time comprised between addition of GO and determining the fluorescence intensity to yield a detection result (GO incubation) may range between 10 and 120 min. In particular embodiments, GO incubation is comprised from 45 to 90 min, or from 60 to 80 min, or from 70 to 80 min. In another particular embodiment GO incubation takes place during 75 min.
  • the qualitative bioassay of the invention may take place in
  • the means may include readily conjugated QD-binding molecule probes.
  • the probes are more than one type of QD-binding molecule probe designed for more than one different analytes.
  • the means may also include a solid support, which in certain embodiments may be readily functionalysed for direct attachment of the QD-binding molecule probes.
  • the solid support has a format selected from microarray format, microtiter plate format and lateral flow format.
  • the kit of the invention comprises a solid support to which the QD-binding molecule probes are already attached.
  • the solid support in addition to the pre-attached probes, the solid support is also blocked for direct use, i.e., ready for addition of the analyte.
  • the solid support pre-loaded with QD-binding molecule probes has a microarray or lateral flow format.
  • the solid support with microarray format contains more than one type of QD-binding molecule probe designed for more than one different analyte. In other embodiments the solid support with microarray format contains more than one QD-binding molecule probe designed for the same analyte.
  • the kit comprises QDs and binding molecules, such that the user may perform the conjugation of the QD- binding molecule probe. In particular embodiments both the QDs and the binding molecules are adequately functionalised for direct conjugation.
  • the means may also include spacer particles.
  • the spacer particles are readily conjugated to at least one binding molecule. In other embodiments spacer particles and binding molecules are provided
  • both the spacer particles and the binding molecules are adequately functionalised for direct
  • the kit provides more than one type of spacer particle-binding molecule probe designed for more than one different analyte.
  • the kit may also comprise adequate buffers and solvents to perform the method of the invention, for example, binding buffer, washing buffer, blocking buffer.
  • the kit may also comprise the reagents required for appropriate functionalisation of the QDs, binding-molecules, spacer particles and/or solid support.
  • the kit comprises a device for point of use fluorescence detection in addition to the probes and reagents for performing the method of the invention.
  • Such point of use fluorometers have reduced dimensions and are known in the state of the art for analyte detection.
  • One example of the point of use/personal devices is commercialised by Promega (see Quantus fluorometer).
  • the kit comprises a device for point of use fluorescence detection and a solid support which comprises a nitrocellulose membrane to which the QD-binding molecule probes are attached, and wherein the assay has lateral-flow format.
  • NH2-functionalized aminosilane glass slides Cat No. 40006 GAPS II, Corning Incorporated, New York, USA
  • silicon slides from Silicon Inc. (Idaho, USA).
  • Anti-E. coli antibodies were from Abeam (ab68451 , Cambridge, UK). Streptavidin-Cy3 and streptavidin-Quantum dot 655 (Cat No.
  • Q10121 MP were obtained from Life Technologies (California, USA). PBS with 2% (v/v) glycerol was used as spotting buffer. PBS supplemented with 5% (w/v) of milk powder and 0.005% (v/v) of Tween 20 was prepared as blocking buffer. PBS supplemented with Tween 20 at 0.05% (v/v) (PBST) was used as washing buffer. PBS with 0.5% (v/v) Tween 20 containing 1 % of BSA fraction V (w/v) was employed as immunobuffer. All aqueous solutions were prepared in Milli-Q water. Escherichia coli O157:H7 (CECT 4783) and
  • Salmonella typhimurium (CECT 722T) strains were obtained from the
  • cell suspensions were prepared directly in 5 mL sterile PBST or tap water, using the bacterial colonies to obtain a bacterial load of 1 .5 x 10 8 CFU/mL-using Densimat apparatus (bioMerieux, Lyon, France). Tubes with the assayed samples were placed into a boiling water bath (100 °C) for 15 min to stop pathogen replication. The tubes were then cooled at room temperature, and finally, refrigerated (at 4 °C) until used.
  • the masked slides were washed with PBST (5 x 100 ⁇ _ per well) and with Milli-Q water (2 x), unmasked, and then dried by centrifugation (1500 rpm x 1 min).
  • the resulting microarray slides were scanned (excitation: 633 nm; emission filter: 670 nm) in a ScanArray 4000 microarray scanner (Perkin Elmer; Massachusetts, USA), and the observed intensity of the probes was taken as the starting intensity.
  • the slides were re-masked, and then incubated with GO (diluted in Milli-Q water at different concentrations from 50 to -350 ⁇ g mL) at two different incubation times (30 or 75 minutes), and then, washed again with Milli-Q water (3 x 150 ⁇ _ per well). Finally, the slides were unmasked, and then dried by centrifugation (1500 rpm x 1 min). The slides were scanned again (excitation: 633 nm; emission filter: 670 nm). The fluorescence intensity of the arrays was quantified using GenePix software (Molecular Devices; California, USA). The fluorescence intensity values were estimated by first measuring the mean intensities of the assayed spots, and then subtracting the local background. The intensity values were normalized by dividing each one by the maximum value of the respective screening.
  • E. coli detection using an immunosandwich configuration Once blocked, the masked slides were washed with PBST (5 x 100 ⁇ _ per well) and the microarrays were incubated for 2 hours with E. coli in PBST (at different concentrations; 100 ⁇ _ per well), washed with PBST (x 3), incubated with Cy3-labelled anti-E. coli antibodies (3 pg/ml_) for 1 hour, and then re-washed with PBST (x 3) and Milli-Q water (x 2). Finally, the slides were unmasked, and then dried by centrifugation (1500 rpm x 1 min). The slides were scanned again (excitation: 543 nm; emission filter: 570 nm).
  • the fluorescence intensity of the arrays was quantified using GenePix software (Molecular Devices; California, USA).
  • the fluorescence intensity values were estimated by first measuring the mean intensities of the assayed spots, and then subtracting the local background. The intensity values were normalized by dividing each one by the maximum value of the respective screening. The limit of detection
  • FIG 2a shows a SEM image of Ab-QD complexes inside a microspot
  • FIG. 2b a SEM image of Ab-QDs/GO complexes inside a microspot.
  • Ab-QD probes were again printed in microspots of ca. 140 ⁇ 10 ⁇ , and then monitored their interaction with E. coli O157:H7 as target pathogen, at different concentrations of E. coli (from 5 to 10 7 CFU/mL), as described in the materials and methods section. Owing to its high density of functional groups, GO can interact with bacteria, resulting in bacterial cell deposition. The pathogen samples were assayed in phosphate-buffered saline (PBS) supplemented with Tween 20 (0.05 %, v/v). The Ab-QD spots were incubated with GO, and subsequently became coated with GO— likely due to
  • PBS phosphate-buffered saline
  • FIG. 2c shows SEM images of E. coli cells (and cell fragments) that were pre-attached (via their membranes) to Ab-
  • the system exhibited a sharp transition at an E. coli concentration of ca. 10 CFU/mL and became saturated at an E. coli concentration of ca. 10 7 CFU/mL (see FIGs. 3 and 5).
  • the probes were scarcely quenched relative to the control signal (i.e. the probes only, incubated with blanks throughout the assay) (see FIG. 3 and FIG. 5).
  • LOD limit of detection
  • the target pathogen was analysed at several concentrations (from 3 to 10 7 CFU/mL) in tap water samples.
  • the system exhibited different quenching levels when PBS was used as matrix. This was probably due to the influence of changes in the microenvironment, which typically affects the fluorescence quantum yield and fluorescence decay behavior of QDs.
  • Important aspects of the microenvironment include the polarity and hydrogen bonding capability of the matrix, and the local viscosity, pH and ionic strength.
  • the influence of the matrix was evidenced in the blank signal: the quenching in tap water was -0.33 units, and in PBS, -0.46 units. Later, in the presence of the target pathogen (E. coli ) the quenching in tap water was typically -0.7 units, and in PBS, -0.8 units (see FIG. 3).
  • the target pathogen E. coli
  • the pathogen- detection system When used with GO, the pathogen- detection system according to the invention is highly sensitive, exhibiting an LOD of ⁇ 5 CFU/mL for E. coli in PBS and in tap water. This configuration might be extended to detect other types of analyte (e.g. cancer cells), perform other tasks (e.g. molecular logic operations) or be applied to other nano-biosystems.
  • analyte e.g. cancer cells
  • tasks e.g. molecular logic operations
  • PBST PBS + Tween 20 at 0,05%).
  • Immuno- Buffer PBS + BSA fraction V (1 %, w/v) + Tween 20 (0,5 % v/v).
  • Blocking buffer PBS + Milk powder (5%, w/v) + Tween 20 (0,005% v/v).
  • Spotting buffer PBS + glycerol (2%, v/v).
  • Gluteraldehyde G5882, Sigma Aldrich
  • Graphene oxide (GO), C/O ratio around 1 , lateral size around 500 nm, monolayer ( Angstron Materials, Ohio, USA). Streptavidin-Quantum dot 655 (Cat No. Q10121 MP) were obtained from Life Technologies (California, USA)
  • Analyte (respective antigen) detection using an immunosandwich configuration with spacer particle the detection of the analyte (antigen) is performed on a solid support (NH2-functionalized aminosilane glass slides), following a procedure similar to that described in example 1 but incorporating the extra step of adding antibody-decorated MSBs as spacer particles.
  • the proceedure is summarised in the following table: Table 1 . Proceedure for detection of analyte when using spacer particle, option 1 .
  • analyte is first incubated with the antibody- decorated QDs that are attached to a solid support and then contacted with the spacer particle.
  • the same results may be achieved by first incubating the analyte with the spacer and then contacting the analyte-spacer conjugate with the antibody-decorated QDs that are attached to a solid support.
  • the latter option is further described below.
  • Option 2 Reagents, instrumentation, functionalization of QD with Ab1 and MSB pre- treatment are the same as above. Functionalization of MSB with Ab2 is also the same up to step 7. Differently from the proceedure above, however, the Ab2-MSB conjugates are not diluted.
  • a method for detecting an analyte of size comprised from 20 to 10000 nm in a mixture which comprises: (i) contacting the analyte with a quantum dot that is conjugated to at least one binding molecule, wherein the binding molecule has affinity for the analyte,
  • step (ii) determining the fluorescence emission intensity of the mixture of step (i) upon exposing the mixture to an electromagnetic radiation capable of exciting the quantum dot
  • step (iv) determining the fluorescence emission intensity of the mixture of step (iii) upon exposing the mixture to the same electromagnetic radiation used in step (ii), wherein a reduction in fluorescence emission intensity of the mixture of step (iii) compared with that of step (i) is indicative of the absence of the analyte.
  • electromagnetic radiation capable of exciting the quantum dot has a wavelength comprised from 360 to 725 nm.
  • (b) contains a lateral size comprised from 5 to 1000 nm
  • (c) contains from 1 to 20 layers, and wherein (d) the final concentration of graphene oxide in the mixture is comprised from 0.01 to 400 pg /ml_.
  • (a) contains an atomic carbon to oxigen ratio (C/O) comprised from 1 to 2,
  • (c) contains from 1 to 20 layers.

Abstract

La présente invention concerne une méthode permettant de détecter un analyte d'une taille allant 1 de 10 000 nm dans un mélange. La méthode comprend les étapes suivantes consistant à : (i) mettre en contact l'analyte avec (a) un point quantique qui est attaché à un support solide et conjugué à au moins une première molécule de liaison, la première molécule de liaison présentant une affinité pour l'analyte, et (b) éventuellement une particule d'espacement conjuguée à au moins une seconde molécule de liaison, la seconde molécule de liaison présentant une affinité pour l'analyte et pouvant être identique ou différente de la première molécule de liaison, et la particule d'espacement possédant une taille allant de 20 nm à 10 000 nm et étant optiquement inactive ; lorsque l'étape (b) est réalisée, les étapes (a) et (b) sont réalisées dans l'importe quel ordre ; (ii) déterminer l'intensité d'émission de fluorescence du mélange de l'étape (i) lorsque le mélange est exposé à un rayonnement électromagnétique capable d'exciter le point quantique ; (iii) ajouter de l'oxyde de graphène au mélange de l'étape (ii) ; et (iv) déterminer l'intensité de l'émission de fluorescence du mélange de l'étape (iii) lorsque le mélange est exposé au même rayonnement électromagnétique que celui utilisé dans l'étape (ii), une réduction d'intensité de l'émission de fluorescence du mélange de l'étape (iv) comparée à celle de l'étape (i) indiquant l'absence de l'analyte ; à condition que lorsque l'analyte a une taille allant de 1 à 19 nm, l'étape (i) (b) doit être réalisée.
PCT/EP2014/072100 2013-10-15 2014-10-15 Dosage biologique qualitatif sensible ay moyen d'oxyde de graphène comme agent révélateur d'analyte WO2015055708A1 (fr)

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CN108525679A (zh) * 2018-05-22 2018-09-14 南开大学 一种以金属有机框架为前驱体制备硫化锌量子点与还原氧化石墨烯复合物的方法
CN109187510A (zh) * 2018-09-05 2019-01-11 浙江理工大学 一种基于电化学发光法检测古代毛织品的方法
CN114674900A (zh) * 2022-04-02 2022-06-28 湖北大学 基于小分子探针的光电化学微传感器及其制备方法与应用
CN115453106A (zh) * 2022-09-22 2022-12-09 山东大学 一种大田软海绵酸免疫检测复合探针及制备方法与应用

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CN105424927A (zh) * 2015-11-09 2016-03-23 山东省海洋生物研究院 一种副溶血弧菌检测方法
CN106290274A (zh) * 2016-07-28 2017-01-04 冯路桥 大肠杆菌石墨烯量子点免疫荧光探针溶液制备及检测方法
CN107561055A (zh) * 2017-08-16 2018-01-09 常州二维碳素科技股份有限公司 一种判定材料中石墨烯成分及含量的方法
CN107561055B (zh) * 2017-08-16 2020-09-01 常州二维碳素科技股份有限公司 一种判定材料中石墨烯成分及含量的方法
CN108525679A (zh) * 2018-05-22 2018-09-14 南开大学 一种以金属有机框架为前驱体制备硫化锌量子点与还原氧化石墨烯复合物的方法
CN108525679B (zh) * 2018-05-22 2020-10-27 南开大学 一种以金属有机框架为前驱体制备硫化锌量子点与还原氧化石墨烯复合物的方法
CN109187510A (zh) * 2018-09-05 2019-01-11 浙江理工大学 一种基于电化学发光法检测古代毛织品的方法
CN109187510B (zh) * 2018-09-05 2020-10-16 浙江理工大学 一种基于电化学发光法检测古代毛织品的方法
CN114674900A (zh) * 2022-04-02 2022-06-28 湖北大学 基于小分子探针的光电化学微传感器及其制备方法与应用
CN114674900B (zh) * 2022-04-02 2023-09-22 湖北大学 基于小分子探针的光电化学微传感器及其制备方法与应用
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