Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
  • A61B10/0045—Devices for taking samples of body liquids

Definitions

• the present invention relates to devices and methods for the enrichment of nucleic acids in vivo and in vitro, particularly to the enrichment and harvesting of circulating nucleic acids such as DNA and RNA from blood or other bodily fluids as well as from extracorporeal environments.
• body fluids comprising, for example, blood, saliva, or urine
• biological or chemical reagents which induce a signal such as luminescence, fluorescence or a change in color specific for the substance to be measured.
• the signal can be registered with the naked eye or with a suitable detection device (i.e. a reader).
• Multiple target substances present in the same sample can be measured in parallel, for example, by aliquoting said sample into separate entities where to each of said entities a reagent specific for the target substance is added, which is subsequently measured.
• said sample may be separated chromatographically, and each of said separated aliquot may be measured directly or may be combined with reagents specific for the target substance to be measured subsequently. Furthermore, reagents discernible in a multiplex readout may be employed. In vitro diagnostics has become an indispensable tool in clinical analysis.
• Fetal cells and fetal DNA that is found in maternal blood can be analyzed in prenatal diagnostics, so that the previously used amniocentesis bearing a high risk of abortion can be avoided.
• Circulating Tumor Cells not only indicate the existence of a tumor, but also provide information about the degree of metastasis and further tumor parameters important for specific further treatment of the patient.
• the enrichment of CTCs is, for example, disclosed in US 2012/0237944 Al .
• This disclosure relates also to a device for isolating CTC directly from blood.
• said device is a gold coated steel wire, which can be introduced into a blood vessel of a human or an animal.
• Capture molecules e.g. antibodies, binding specifically to CTC can be attached to the gold surface. Once inserted into the blood stream, the number of CTC bound to capture molecules is constantly increasing. After retracting the steel wire, CTC can be isolated and analyzed.
• This method delivers off-line signals, i.e. after retraction of the steel wire from the body.
• nucleic acid molecules are present in organisms or in other environments in only minute quantities, and considering the fact that usually only small amounts of liquid biopsy material or small samples can be retrieved from an organism (e.g. most frequently blood samples from humans are taken in amounts of 5 ml or 10 ml for further analysis), there is a considerable likelihood that the amounts of nucleic acids present in an organism (or other environments) cannot be detected using in vitro analysis methods, such as PCR, immunoassays, etc..
• nucleic acids of interest it would also be desirable to be able to measure both, nucleic acids of interest at a given moment or over time.
• the latter measurements could be used in quantifying kinetics or in the initiation of alerts once threshold values are exceeded or fall short of certain threshold values.
• Such methods and respective means could be extremely helpful in the detection, analysis and/or quantification of nucleic acids derived, e.g. from pathogens such as microorganisms or from cancer cells.
• pathogens such as microorganisms or from cancer cells.
• such means and methods would also comprise the identification of pathogens at the level of strains as well as the determination of antibiotic resistances.
• None of the known devices and/or methods solves the problem of collecting and of enriching of nucleic acids of interest at the surfaces of the device in situ, e.g. whilst it is inserted in a blood vessel or any other corporeal or extracorporeal environment potentially comprising such nucleic acids, and analyzing said nucleic acids of interest either online without the risk of shedding fibre components which would inevitably lead to complications for the patient or subsequently in vitro
• devices for the enrichment or analysis of nucleic acids of interest after removal of a device from an organism in vitro, in particular from patients are not known. Still further, it was not possible to enrich nucleic acids of interest using devices to be introduced in vivo into an organism, removing the same from said organism, analyzing bound nucleic acids of interest, e.g. in order to identify pathogens or nucleic acid molecules derived therefrom, and to use (RT-)PCR-based amplification or isothermal amplification methods and/or other nucleic acid analysis techniques (for example Next Generation Sequencing).
• nucleic acids of interest may be analyzed directly and/or online (i.e. while the probe of said device is inserted in the individual to be analyzed) in blood vessels or other body cavities, especially those with flowing body fluids.
• the device comprising a probe is suitable for the enrichment of nucleic acids of interest in vivo (in corporeal environments) and in extracorporeal environments.
• bound nucleic acids of interest can be analyzed after removal of the probe from the corporeal or extracorporeal environment and to analyze enriched nucleic acids in vitro, e.g. using molecular biology methods, immunological methods or other procedures, such as MALDI.
• Fig. 1 shows a fibre for the enrichment of nucleic acids of interest set up as contemplated in embodiments of the invention.
• a fibre is used with a core made of glass fibre.
• a jacket (101) protects the fibre from mechanical damage.
• a coating (113) made of covering the core at least partly to enable for enrichment and optionally also for optical measurement technologies like SPR or SERS or to protect the exposed core mechanically.
• Core, coating, cladding or jacket may further be covered by a second coating layer (115), usually employed to limit unspecific interaction of the surrounding fluids with the fibre, or to improve the attachment of capture elements to the fibre.
• Capture elements like single stranded nucleic molecules or specific anti-nucleic acid molecules, or aptamers, Peptide Nucleic Acids (PNA) and derivatives thereof may be attached directly or by a linker to surfaces of the fibre. Different ways to attach capture elements may have been performed with the same fibre. For example, the capture element (118) is directly attached to the core (103) of the fibre, capture element (117) to the second coating (115), and capture element (1 14) to the first coating (113).
• the fibre may comprise further coatings (109) in other areas of the exposed fibre surfaces, e.g. coatings of different material or thickness compared to coating (113).
• Different capture elements (111) - with or without linker (110) may be attached in different areas, targeted to different target nucleic acids of interest, e.g. to those derived from bacteria (112), viruses, fungi, parasites, or from eukaryotic cells.
• the tip of the fibre may be equipped with another coating or optical element (104), i.e. cone, DOE, mirror or filter, which may further be structured for particular optical techniques including, i.e., (attenuated) total reflection.
• the tip may carry further capture elements (106).
• Capture elements (106, 1 14, 1 17, 118, or 11 1) may comprise further, in particular optically active elements (108), i.e. fluorescent dyes.
• the cladding (102) is omitted, the remainder of the figure would be identical.
• Fibre bundles may comprise the same type of fibres (201), or different sizes or types of fibres (203). Fibres may e.g. be fused or glued (202). Gaps between fibres may exist, either filled with material (204) or not. Other functional elements (205) may be combined with fibres, i.e. conducting elements.
• Figure 3 shows an embodiment of the invention, in which the fibre is protected by an encasing sheath (301).
• the sheath may be moved relative to the fibre as indicated by the double arrow.
• a diaphragm (302) or protective cap may be removed or broken by the movement of the fibre or sheath, or by another active mechanism.
• Figure 4 shows the results of qPCR analysis of different concentrations of E. coli added to PBS and enriched using an enrichment device having (A) a gold-coated surface and (B) a polymer surface. It can be seen that the qPCR-based detection of E. coli was possible after enrichment of the bacteria from PBS spiked with 10 6 to 10 2 CFU/ml of E. coli strain K12.
• FIG. 5 shows the results of qPCR analysis of different concentrations of E. coli added to whole blood derived from human blood reserves. Enrichment was performed with an enrichment probe having a gold-coated surface, either (A) by dipping the probe into human blood spiked with E. coli in a tube, or (B) by introduction of said enrichment probe into an artificial blood circulation system comprising PBS spiked with E. coli. It can be seen that the qPCR-based detection of E. coli was possible after enrichment of the bacteria from PBS spiked with 10 6 to 10 2 CFU/ml of E. coli strain K12 transformed with Green Fluorescent Protein (GFP)).
• GFP Green Fluorescent Protein
• Figure 6 shows the result of a qPCR-based detection of E. coli enriched in vivo for 15 min from a rat inoculated with E. coli.
• Figure 7 shows an agar plate with colonies of E. coli formed within 12 hours after inoculation by inoculating the agar surface with an enrichment probe inserted for 15 min into a rat previously inoculated with E. coli or not.
• the left side of the petri dish (labeled "Probe 1") was scratched with an enrichment probe that was inserted for 15 min into a rat that was not inoculated with E. coli and the right side (labeled "Probe 2) shows colonies formed after scratching the agar with an enrichment probe inserted into a rat previously inoculated with E. coli.
• Figure 8 shows an agar plate with colonies of S. aureus formed within 12 hours after inoculation by inoculating the agar surface with an enrichment probe inserted for 30 minutes into human blood into an artificial blood flow system spiked with S. aureus.
• A shows colonies grown on agar inoculated with S. aureus by scratching the agar with the enrichment probe and
• B shows colony growth around a piece of an enrichment probe cut off from said device and placed onto an agar plate.
• the terms “about” and “approximately” denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question.
• the term typically indicates a deviation from the indicated numerical value of ⁇ 20 %, preferably ⁇ 15 %, more preferably ⁇ 10 %, and even more preferably ⁇ 5 %.
• first”, “second”, “third” or “(a)”, “(b)”, “(c)”, “(d)” etc. relate to steps of a method or use there is no time or time interval coherence between the steps, i.e. the steps may be carried out simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in the application as set forth herein above or below.
• a “polynucleotide”, “nucleic acid” or “nucleic acids of interest” is a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribo nucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA and RNA including also classes of nucleic acids that seem to have regulatory function, e.g. miRNA.
• a typical "antibody”, which is one type of capture molecule according to the present invention, comprises a tetramer of polypeptides.
• Each tetramer is composed of two pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
• the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
• the terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.
• the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.
• Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chains.
• Heavy chains are classified as mu ( ⁇ ), delta ( ⁇ ), gamma ( ⁇ ), alpha (a), and epsilon ( ⁇ ), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
• Several of these may be further divided into subclasses or isotypes, e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2.
• Different isotypes have different effector functions; for example, IgGl and IgG3 isotypes have antibody- dependent cellular cytotoxicity (ADCC) activity.
• Human light chains are classified as kappa ( ⁇ ) and lambda ( ⁇ ) light chains.
• variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids.
• J Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)).
• Allotypes are variations in antibody sequence, often in the constant region, that can be immunogenic and are encoded by specific alleles in humans. Allotypes have been identified for five of the human IGHC genes, the IGHG1, IGHG2, IGHG3, IGHA2 and IGHE genes, and are designated as Glm, G2m, G3m, A2m, and Em allotypes, respectively.
• IGHC genes the IGHG1, IGHG2, IGHG3, IGHA2 and IGHE genes
• V, D, J and constant (C) gene segments Prior to the rearranging and joining of various immunoglobulin gene segments, the V, D, J and constant (C) gene segments are found generally in relatively close proximity on a single chromosome. During B-cell-differentiation, one of each of the appropriate family members of the V, D, J (or only V and J in the case of light chain genes) gene segments are recombined to form functionally rearranged variable regions of the heavy and light immunoglobulin genes. This gene segment rearrangement process appears to be sequential. First, heavy chain D-to- J joints are made, followed by heavy chain V-to-DJ joints and light chain V-to-J joints.
• antibody is used in the broadest sense and includes fully assembled antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (including bispecific antibodies), antibody fragments that can bind an antigen (including, Fab', F'(ab)2, Fv, single chain antibodies, diabodies), and recombinant peptides comprising the foregoing as long as they exhibit the desired biological activity. Multimers or aggregates of intact molecules and/or fragments, including chemically derivatized antibodies, are contemplated.
• Antibodies of any isotype class or subclass including IgG, IgM, IgD, IgA, and IgE, IgGl, IgG2, IgG3, IgG4, IgAl and IgA2, or any allotype, are contemplated.
• hypervariable region refers to amino acid residues from a complementarity determining region or CDR (i.e., residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain as described by Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Even a single CDR may recognize and bind antigen, although with a lower affinity than the entire antigen binding site containing all of the CDRs.
• CDR complementarity determining region
• Antibody fragments comprise a portion of an intact immunoglobulin, e.g., an antigen binding or variable region of the intact antibody, and include multispecific (bispecific, trispecific, etc.) antibodies formed from antibody fragments. Fragments of immunoglobulins may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
• Non- limiting examples of antibody fragments include Fab, Fab', F(ab')2, Fv (variable region), domain antibodies (dAb, containing a VH domain; Ward et al, Nature, 341, 544-546, 1989), complementarity determining region (CDR) fragments, single-chain antibodies (scFv, containing VH and VL domains on a single polypeptide chain) (Bird et al, Science, 242:423-426, 1988, and Huston et al, Proc. Natl. Acad. Sci., USA 85:5879-5883, 1988, optionally including a polypeptide linker; and optionally multispecific, Gruber et al, J.
• VHH containing antibody a VHH containing antibody
• heavy chain antibodies HCAbs, homodimers of two heavy chains having the structure H2L2
• polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide, such as a CDR sequence, as long as the antibody retains the desired biological activity.
• monoclonal antibody refers to an antibody, as that term is defined herein, obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations or alternative post-translational modifications that may be present in minor amounts, whether produced from hybridomas or recombinant DNA techniques.
• Non-limiting examples of monoclonal antibodies include murine, rabbit, rat, chicken, chimeric, humanized, or human antibodies, fully assembled antibodies, multispecific antibodies (including bispecific antibodies), antibody fragments that can bind an antigen (including, Fab', F'(ab)2, Fv, single chain antibodies, diabodies), maxibodies, nanobodies, and recombinant peptides comprising the foregoing as long as they exhibit the desired biological activity, or variants or derivatives thereof.
• Humanizing or modifying antibody sequence to be more human-like is described in, e.g., Jones et al, Nature 321 :522 525 (1986); Morrison et al, Proc. Natl. Acad.
• Phage display is described in e.g., Dower et al, WO 91/17271 , McCafferty et al, WO 92/01047, and Caton and Koprowski, Proc. Natl. Acad. Sci. USA, 87:6450-6454 (1990), each of which is incorporated herein by reference in its entirety.
• Another method for isolating human monoclonal antibodies uses transgenic animals that have no endogenous immunoglobulin production and are engineered to contain human immunoglobulin loci. See, e.g., Jakobovits et al, Proc. Natl. Acad. Sci.
• markers for health and/or disease in living organisms e.g. patients
• markers for possible presence and/or absence of contaminants in an organism or extracorporeal environment may, for example, be a sepsis, an infection, cancer, autoimmune disease for which marker nucleic acids may be present, etc.
• markers for health or disease can be some fetal-derived nucleic acids of interest in maternal blood, e.g. those confirming a pregnancy, the absence or presence of a mutation that is associated with disease or well-being of a fetus.
• Nucleic acids may also be derived from an organism whose health or disease status may be checked, e.g. for the presence of nucleic acid transcripts usually associated with certain disease conditions (e.g. nucleic acids indicative of an ongoing immune response or with certain autoimmune diseases) including also disease-associated miRNA molecules or miRNA that are often ubiquitously found in individuals that are, or appear to be, healthy.
• certain disease conditions e.g. nucleic acids indicative of an ongoing immune response or with certain autoimmune diseases
• the quantity of the nucleic acids of interest can be determined and compared to a threshold level using statistical methods in order to confirm statistically significant or insignificant deviations from levels in controls (e.g. healthy organisms or organisms with confirmed disease).
• the presence or absence and/or the quantity of the nucleic acids of interest may be correlated in inventive methods aiming at prognosticating the occurrence and/or development of disease, e.g. a deterioration or amelioration.
• An example is a method of prognosticating the development of cancer and the likelihood and expected time of survival using certain markers, e.g. mutations that correlate favorably or to the detriment of a patient.
• certain markers e.g. mutations that correlate favorably or to the detriment of a patient.
• some mutations in the egfr or kras genes influence the susceptibility of a cancer cell to a treatment, e.g. using certain tyrosine kinase inhibitors.
• the present invention also relates to methods of prognosticating the disease development using the herein below described means and methods for the enrichment and analysis of certain nucleic acids of interest.
• Epitope or “antigenic determinant” refers to a site on an antigen present on nucleic acids of interest to which an antibody binds. Epitopes can be formed both polynucleic acids juxtaposed by tertiary folding.
• the specified capture molecules such as antibodies, bind to a particular epitope at least two times the background and more typically more than 10 to 100 times background.
• the terms “capturing”, “binding”, “attaching” refer to a specific interaction between capture molecule and target, permitting the enrichment and subsequent analysis of the bound target, e.g. the detection, identification and/or the analysis using cell culture, molecular genetics, physical or chemical characterization, and i.e. the description of structural or functional properties of such target.
• binding affinity refers to the equilibrium dissociation constant (K D ) associated with each antigen-antibody interaction.
• the antibodies described herein exhibit desirable properties such as binding affinity as measured by K D for a target in the range of 1 x 10 ⁇ 6 M or less, or ranging down to 10 "16 M or lower, (e.g., about 10 "6 , 10 “7 ,10 “8 , 10 “9 , 10 “10 , 10 11 , 10 "12 , 10 “13 , 10 “14 , 10 “15 , 10 ⁇ 16 M or less) at about pH 7.4, where lower KD indicates better affinity.
• the equilibrium dissociation constant can be determined in solution equilibrium assay using, e.g., BIAcore.
• the binding affinity is directly related to the ratio of the kinetic off-rate (generally reported in units of inverse time, e.g. seconds "1 ) divided by the kinetic on-rate (generally reported in units of concentration per unit time, e.g. M/s).
• Off-rate analysis can estimate the interaction that occurs in vivo, since a slow off-rate would predict a greater degree of interaction over a long period of time.
• hybridization refers to the specific interaction between a capture molecule and a nucleic acid of interest.
• Specific hybridization means that nucleic acids used as capture molecules hybridize with complementary strands in the corporeal or extracorporeal environment conditions to which the probe of the device according to the present invention is exposed or that is brought into contact with said probe.
• the hybridized nucleic acids i.e. capture nucleic acid and nucleic acid of interest
• At least regions of the targeted nucleic acid of interest having the same length as the capture nucleic acid are at least 70% complementary, at least 75% complementary, at least 80% complementary, at least 85% complementary, at least 90% complementary, at least 95% complementary, at least 96% complementary, at least 97% complementary, at least 98% complementary, or at least 99% complementary.
• the capture molecules can be short (e.g. only 5, 6, 7, 8, 9, 10, 15, 20, 25 nucleotides long - or any length between two of these values, e.g. 13, 18, or 23) or longer (e.g.
• target relates to nucleic acids of interest (NAI or nai) derived from the individual or environment into which the sampling device or probe according to the present invention was introduced, or to nucleic acids that a not generally found in such individuals or environments, e.g. nucleic acids derived from microorganims such as bacteria, fungi, parasites or viruses, nucleic acids released from (dead) cells, e.g. from cancer cells, e.g. those destroyed by immune cells or under the influence of chemical compounds
• the target nucleic acids are derived from bacteria, fungi or viruses.
• the nucleic acids of interest are derived from bacteria, fungi or viruses, which may cause sepsis, or are markers of infection with a specific or non-specific pathogen.
• the targets bound to the probes used in context of the present invention may be detached using mechanical, physical, chemical, and enzymatic methods.
• Sepsis refers to an inflammatory condition caused by an infection with a microorganism, e.g. bacteria, parasites, viruses or fungi as known to person skilled in the art. Sepsis includes system inflammatory response syndrome (SIRS), wherein an individual shows a number of clinical signs, e.g. an increased or decreased body temperature (>38°C or ⁇ 36°C), elevated heart frequency (>90 beats/min), and breathing frequency, increased or decreased numbers in leukocytes (>12.000 mm3 or ⁇ 4.000 mm3), hypotonia and reduced perfusion, and organ dysfunction.
• SIRS system inflammatory response syndrome
• microorganism inducing sepsis relates to any microorganism that causes an infection that may induce blood-poisoning comprising, inter alia, staphyloccoci including Methicillin Resistant Staphylococcus Aureus (MRSA), streptococci, gram negative gastrointestinal bacteria such as E.
• staphyloccoci including Methicillin Resistant Staphylococcus Aureus (MRSA), streptococci, gram negative gastrointestinal bacteria such as E.
• coli Klebsiella, Enterobacter, Proteus, Pseudomonas aeruginosa, Bacteroides, or Menigococci, Haemophilus influenzae, Clostridiae, Listeriae, Salmonellae, Pasteurella multocida, Gonococci, Aeromonas, Campylobacter, Serratia marcescens, coagulase-negative Salmonellae, Acinetobacter species, Pseudomonas species, Bacillus cereus, fungi such as Candida, Aspergillus, etc., Dengue viruses, Herpes viruses.
• virus refers to any virus and includes important human pathogens, e.g. HIV or other Retroviridae, Hepadnaviridae, Herpesviridae, Noroviruses, Flaviviridae, Bunyaviridae, Filoviridae, Picornaviridae, Papillomaviridae, Adenoviridae, Plant viruses, animal viruses, particularly viruses capable of infecting domestic animals and livestock, etc., or those infecting crop plants, ornamental plants, etc..
• HIV or other Retroviridae Hepadnaviridae, Herpesviridae, Noroviruses, Flaviviridae, Bunyaviridae, Filoviridae, Picornaviridae, Papillomaviridae, Adenoviridae
• Plant viruses animal viruses, particularly viruses capable of infecting domestic animals and livestock, etc., or those infecting crop plants, ornamental plants, etc.
• fungus refers to any known human, animal or plant pathogen, e.g. Saccharomyces, Candida, Cyrptococcus, Geotrichum, Rhodotorula, Sporobolomyces, Trichosporon, Epidermophyten, Mikrosproium, Piedraia, Trichophyton, Aspergillus, Mucor, etc.
• probe and “sampling probe” designates the part of the sampling device that is at least partially introduced into a sample container, a tube, an environment of interest, or an individual whereof at least one clinical parameter should be determined, a marker should be identified, or wherein an infection should be diagnosed, etc.
• the term "environment” designates any extracorporeal liquid environment that may contain nucleic acids of interest, e.g. any fluid found in hospitals, in refectories, in the food and drink industry, in public facilities, in sewage channels, in drinking water reservoirs, fountains, but also in cell cultures, nutrients, beverages, etc.
• fibre can designate both, optically transparent fibres for respective optical measurements of enriched target(s), e.g. nucleic acids of interest, and fibres that are not used for optical analysis of enriched target(s). Both form part of the probe of the present invention that is at least partially introduced into an individual referred to above.
• Optical fibres usually comprise a core at the center of the fibre and a sheath surrounding the core which is called cladding. Both, core and cladding are optically transparent, and the refractive index of the core is higher than the refractive index of the cladding.
• cladding a sheath surrounding the core
• refractive index of the core is higher than the refractive index of the cladding.
• “Step index” fibres are provided with an abrupt interface between core and cladding, and consequently an abrupt decrease of the refractive index at the interface from core to cladding. Light propagating through the fibre core is reflected at this interface and will thus not leave the core unless the fibre is strongly bent.
• "Gradient index” fibres exhibit a smooth decrease of the refractive index from the fibre axis to the outer surface, which also impedes light leaving the fibre.
• fibres may be manufactured of homogeneous material, but may contain hollow hoses, which alter the refractive properties of the fibers. These fibres are also called photonic crystal fibres. A jacket often made of plastic material usually protects fibres. In most cases it sheathes the fibre surface. Fibres may carry capture elements that are either directly or indirectly coupled to their surface in regions with no jacket present.
• the fibres are glass fibres coated with a polymer layer.
• Said polymer layer may be functionalized with molecules that serve as anchors for the capture molecules.
• the polymer-coated fibres are either not coated with metals, particularly not with noble metals such as gold or platinum, which makes them less expensive and their production much easier, or they are coated with such metals.
• Plastic fibres or PMMA fibres are even more rigid; they can be bent, but do not break. Further, these fibres are considerably less expensive than other carrier materials. Further, the fibres may also be metal-coated polymeric fibres, e.g., polymeric fibres coated with metals selected from the group comprising gold, platinum, silver, copper, nickel, etc., and optionally, polymeric fibres, e.g. glass fibres that comprise a metal-coating such as an aluminium coating that is further at least partially coated with gold.
• metal-coated polymeric fibres e.g., polymeric fibres coated with metals selected from the group comprising gold, platinum, silver, copper, nickel, etc.
• polymeric fibres e.g. glass fibres that comprise a metal-coating such as an aluminium coating that is further at least partially coated with gold.
• fibre surface denotes any boundary interface of the (optical) material towards air or fluid.
• the term may also denote the polymer-coating of a glass fibre to which capture molecules are attached or bound and that is exposed to the environment, e.g. the bodily fluid of a patient. This term never denotes the surface of the fibre jacket.
• antibiotic compound means an agent that either kills or inhibits the growth of a microorganism.
• antifungal compound and “antiviral compound” relate to agents that inhibit the growth or reproduction of fungi or viruses. These compounds or agents may be summarized as "microbicides”.
• capture elements comprises molecules, for example antibodies, peptides, or aptamers, which are attached to the surface of an optical, e.g. silver halide fibre, or to polymer-coated glass fibre, and which are capable of binding specifically to other molecules, e.g. nucleic acids of interest.
• the molecules may be modified chemically to optimize their biochemical parameters, and may comprise further molecular elements like linkers, chromophores or fluorophores.
• Further examples for molecular capture elements comprise proteins, for example mannose binding lectin and its engineered forms (Kang et al. 2014) or other lectins. They may be concatenated or linked to polymers to increase avidity.
• Capture elements further comprise particles or viruses, the latter bearing capture proteins or peptides within the viral capsid.
• the antibodies bound to the polymer- coated fibres according to the present invention are specific for nucleic acids of interest derived from sepsis-inducing microorganisms, and preferably these antibodies are humanized antibodies.
• the capture molecules may be immobilized using molecules having functional groups, wherein these molecules are layered on the fibre surface and to attach capture molecules to such layer.
• fibres may comprise calcium ions, which bind to phosphate backbones of polynucleic acids via ionic interactions, wherein the binding occurs rather non-specifically.
• the fibres can be glass fibres that are coated with polymeric coatings selected from the group comprising polyimides, polyamides, polyacrylates, or the like.
• Device comprises devices used in a clinical setting and especially devices being in contact with patients and in particular devices being temporarily or permanently in contact with body fluids like blood, serum, lymph, etc., which will be inserted into the body.
• Devices encompass also any apparatus that can be physically connected with the sampling probe according to the present invention as well as kits of parts comprising the sampling probe of the present invention and which may be assembled to permit the methods of the present invention.
• microorganism comprises single cell organisms such a bacteria, fungi, parasites, or viruses. Microorganisms may be found in body fluids in a free form, i.e. as individual cells, or in aggregates, e.g. films, or multiple cocci. Floating microorganisms are also called "planktonic".
• the term "individual” comprises both, healthy individuals and patients with confirmed disease.
• the term further comprises human and veterinary patients, e.g. mammalian patients.
• Individuals may be those suspected to have been exposed to certain nucleic acids of interest of interest, e.g. infectious microorganisms (bacteria, fungi, viruses, etc.), or those that are known to have been exposed to certain nucleic acids of interest, but which do not have any clinical symptoms and/or which do not yet have a confirmed diagnosis (e.g. by qPCR).
• Individuals may also be those with confirmed disease, i.e. those that have clinical symptoms, or those with a confirmed diagnosis for a respective target (e.g.
• patient as used herein comprises humans infected by bacteria, fungi, or viruses, or occasionally humans prone to such infection, regardless of their gender, age, genomic profile, ethnic or anamnesis. It also includes all animals infected, especially domestic animals, such as farm animals including birds and fish, and companion animals like dogs and cats.
• subject/individual at risk of development of a sepsis refers to organisms exposed or at risk of being exposed to sepsis-inducing organisms or molecules derived therefrom.
• immunocompromised patients e.g. patients in a unit for neonates, patients in a pediatric unit, patients known or suspected to have been in contact with other individuals or patients that are known as or suspected to be carriers of sepsis-inducing cells or molecules (e.g. relatives of patients with sepsis, medical staff exposed to patients with sepsis, medical staff working in an environment in which sepsis cases have occurred, etc.), in particular patients suffering from sepsis or those having suffered from a sepsis.
• sepsis-inducing cells or molecules e.g. relatives of patients with sepsis, medical staff exposed to patients with sepsis, medical staff working in an environment in which sepsis cases have occurred, etc.
• silver halide fibre comprises fibres made of AgCkAgBr, where the fibre core contains more bromine than the cladding in step index fibres or the surface region in gradient index fibres, so that the refractive index in the core is higher than in the cladding.
• a synonymous term in literature is polycrystalline infrared or PIR fibre.
• silver halide fibres are manufactured as step index fibres, but gradient fibres also exist.
• Silver halide fibres are polycrystalline and thus consist of grains. Typically the grain size is in the range of 0.1 to 1 ⁇ while the core grains are larger than the cladding or surface grains.
• the term “style”) designates biologic and synthetic polymers that are capable of coating a glass fibre, e.g. a quartz glass fibre coated with synthetic polymers such as those selected from the group comprising polyamine, polyamide, polyimide, polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyurethane, polyacrylic, polyether ketones coatings and the like. It is possible to mix and combine more than one of the synthetic polymers for the purposes of the invention. These polymers can be three-dimensionally immobilized on the glass fibre, e.g. using photografting techniques. Using such photografting techniques, polymer chains are attached on the fibres and thereby form a three-dimensional meshwork of polymers (e.g.
• Photografting comprises techniques designated "photografting from” and/or redesignphotografting-to" as known in the art, i.e. a covalent incorporation of functional additives to a polymer matrix or polymer surface using a light-induced mechanism.
• the length of the polymeric additives depends on the length of light exposure, and the degree of branching can be influenced by addition of cross-linkers.
• the likelihood of a larger number of bound nucleic acids of interest increases with the number of capture probes.
• the fibres are fully polymeric
• synthetic polymers are polyamine, polyamide, polyimide, polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyurethane, polyacrylate, polyethylene ketones, as well as combinations thereof, etc., but they may also be hydrogels, including hydrogels for medical use, Hydrogel with AMPS, or any network of polymer chains that are hydrophilic and permit the attachment of capture molecules.
• MALDI and "MALDI-TOF” designate well established mass spectrometric techniques known to persons skilled in the art.
• MALDI/TOF spectra are used for the identification of microorganisms such as bacteria or fungi. A colony of the microbe in question is smeared directly on the sample target and overlaid with matrix.
• the mass spectra generated are analyzed by dedicated software and compared with stored profiles. Species diagnosis by this procedure is generally considered much faster, more accurate and cheaper than other procedures based on immunological or biochemical tests.
• antibiotic resistance means that a microorganism (bacterium, virus, fungus, or a multicellular parasite) has developed a resistance, it means that a given antimicrobial compound no longer affects metabolic or survival-protecting activities of said microorganism (e.g. spore formation).
• a given microorganism in presence (and/or sufficient quantity) of said compound a given microorganism is still capable of dividing (proliferation), it can also exert metabolic functions ensuring its survival and capability to divide or to sporulate, it may still release factors that are negatively influencing the health, well-being or survival of the host (i.e. the organism carrying said bacterium or the capability to exert normal metabolic functions, in case of viruses, it may replicate its genome, assemble in the infected host cell or the at the cell membrane to form infectious progeny virions, be released from the host cell, interact with cellular compounds such as nucleic acids, enzymes, cell membranes, etc.
• microbicide e.g. antibiotic
• those microorganisms survive and further proliferate, which have developed means to escape from toxic influences exerted by respective microbicide compounds. In other words, they have become resistant. Frequently, such resistant microorganisms are, or become, capable of severely affecting the health of the infected host.
• microbicide resistance concerns the complete or intermediate resistance to at least one or more microbicide compounds known in the art.
• antibiotic or "bacterium/bacterial”
• fungicides e.g. fungicides, virocides, etc.
• virocides e.g. fungicides, virocides, etc.
• a device for capturing, enrichment, detection, identification and/or analysis of a nucleic acid of interest characterized in that it comprises a sampling probe comprising capture elements specific for said nucleic acid of interest.
• the device for capturing, enrichment, detection, identification and/or analysis of a nucleic acid of interest comprises a sampling probe comprising capture elements specific for said nucleic acids of interest, wherein the probe is suitable for use in vivo in a subject, preferably a subject suspected to be at risk of development of a sepsis.
• the device for capturing, enrichment, detection, identification and/or analysis of a target, particularly of a nucleic acid of interest referred to above comprises a sampling probe comprising capture elements specific for said nucleic acid of interest, wherein the probe comprises a glass fibre, fiberglass-enforced plastic fibre, PMMA fibre, metal coated glass fibre, or the like.
• the device comprises a glass fibre comprising a polymer coating, preferably comprising at least one polymer selected from the group comprising synthetic or biological polymers, particularly hydrogels, polyethylene (PE), polypropylene (PP), polyethyleneterephtalat (PET), polyvinylchloride (PVC), polycarbonate (PC), polystyrene, polyamine, polyamide (PA), polytetrafiuorethylene (PTFE), polymethylmethacrylate (PMMA), polyurethane (PUR), polysiloxane, polyetherketone (PEEK), polysulfone (PSU), polyhydroxyethylmethacrylate (PHEMA), polyimide (PI) or combinations thereof, or the fibre consists fully of polymers selected from the above compounds.
• a polymer coating preferably comprising at least one polymer selected from the group comprising synthetic or biological polymers, particularly hydrogels, polyethylene (PE), polypropylene (PP), polyethyleneterephtalat (PET), polyvinylchlor
• the polymer coating may further be coated with a metal such as aluminium that may also be coated,e.g.with a gold-coating.
• the polymer-coating can be a two- or three-dimensional coating obtained by photografting techniques, wherein polymers are immobilized on the probe/ fibre. The longer the polymer chains, the more flexible is the polymer-coating and the higher is the number of functional groups to which capture probes can be attached or bound.
• the device according to any one of the preceding paragraphs with a nucleic acid of interest attached to or a capture molecule is analyzed using a method selected from the group comprising mass spectrometry methods, Surface Plasmon Resonance methods, RAMAN spectroscopy, Infrared Spectroscopy, fluorescence-based methods, ATR, ELISA, molecular biologic methods, in particular (RT)- PCR or sequencing methods (e.g. Next Generation Sequencing.
• the sampling device comprises a probe comprising a fibre, wherein the diameter of the fibre is ⁇ _1 mm.
• the sampling device comprises at least two or more different specific capture elements a 1 , a 2 ,...a n are disposed on the surface of the fibre, wherein said at least two of more different specific capture elements have different specificities for different nucleic acids of interest structures tNAI1 , tNAI2 ,... tNAIn .
• the sampling device as described in any of the preceding embodiments, wherein at least two or more different specific capture elements are disposed on the surface of the fibre at different regions.
• At least two or more different specific capture elements are disposed on the surface of at least two or more different fibres, optionally at different regions of said two or more different fibres.
• the capture elements are selected from the group comprising amino acids, Peptide Nucleic Acids, antibodies, nucleic acids, small molecules, chelating agents binding targets due to ionic interactions, and aptamers.
• the probe is suitable for insertion into the body of an individual or into any environment known or suspected to contain nucleic acids of interests, wherein said individual or environment is selected from the group comprising:
• nucleic acid of interest may further be selected from the group comprising those derived from bacteria, fungi, viruses, parasites, and toxins, particularly those capable of inducing for a sepsis;
• nucleic acid of interest of interest selected from the group comprising those derived from specific bacterial cells, fungal cells, virus particles parasites o;
• patients comprising human patients or veterinary patients;
• extracorporeal liquid environments known or suspected to contain nucleic acids of interests e.g. devices in hospitals such as sterile water sources, nutrient solutions, drips, or liquid environments in restaurants, cantines, and the like, or in waste water or drinking water reservoirs, in breweries, in industrial plants for the manufacturing of medicaments or food and drinks, or in the cosmetics industry, and
• the probe can be used in methods of enriching or capturing as well as analyzing directly of subsequent to removal of nucleic acids from the probe.
• the specific capture elements selectively bind nucleic acids derived from bacterial cells or molecules derived from such cells.
• the specific capture elements selectively bind nucleic acids derived from fungal cells or molecules derived from such cells.
• the specific capture elements selectively bind nucleic acids derived from virus particles or molecules derived from such cells.
• the specific capture elements selectively bind bacterial cells capable of inducing sepsis in an individual or molecules derived from such bacterial cells.
• the capture elements selectively bind bacteria selected from the group of Gram-positive and/or Gram-negative bacteria.
• the capture elements selectively bind bacteria selected from the group comprising staphyloccoci including Methicillin Resistant Staphylococcus Aureus (MRS A), streptococci, gram negative gastrointestinal bacteria, E.
• staphyloccoci including Methicillin Resistant Staphylococcus Aureus (MRS A), streptococci, gram negative gastrointestinal bacteria, E.
• the nucleic acids are derived from bacteria to detect an infection, e.g. an infection with a bacterium involved in the development of sepsis, and further the methods of the invention can include steps aiming at determining whether or not nucleic acids of potentially present bacteria carry mutations that render them resistant against antibacterial treatment.
• nucleic acid of interest indicating the presence or absence of a disease, disorder or medical indication, or a nucleic acid of interest the presence or absence of which is implicated in the development of such disease, disorder or medical condition or a nucleic acid of interest indicating the presence or absence of an effect of a therapeutic agent, in particular therapeutic molecule or its metabolites or degradation products, comprising using the sampling device according to any of the preceding embodiments, further comprising quantifying the specific nucleic acids of interest, and optionally comparing the quantity with a threshold value.
• specific nucleic acids of interests are enriched.
• the specific nucleic acids of interests are derived from bacterial cells, particularly those causing sepsis, optionally selected from the microorganisms referred to above.
• the sampling probe is introduced into an individual.
• an in vitro method for the detection, analysis and/or quantification of specific nucleic acids of interest in an enriched sample obtained from a patient wherein the quantity of obtained nucleic acids of interest bound to a probe as defined in any one of preceding embodiments is determined, optionally preceded by at least one washing step, further optionally comprising comparing the results of said detection, analysis and/or quantification method with threshold value(s) or reference value(s).
• nucleic acids of interest in an enriched sample are characterized using at least one method selected from the group comprising:
• nucleic acids e.g. PCR, RT-PCR and NGS
• Mass spectroscopic methods comprising MALDI-ToF or LC-MS.
• an in vitro method according to any the preceding embodiments is provided, wherein said method is followed by a methods of determining resistance to microbicide compounds, e.g. antibiotics, wherein the enriched nucleic acids of interest may be analysed using any known method to detect resistances to said compounds using molecular genetic procedures, e.g. PCR, qPCR, RT-PCR, high throughput sequencing (also known as Next Generation Sequencing, NGS).
• the captured or enriched nucleic acids of interest are analysed by PCR.
• the DNA or RNA can be removed from the probe or genetic information associated with the development of resistances to microbicides, particularly antibiotics, is amplified.
• PCR primers can be designed to amplify only those specific genetic regions associated with development of resistance to microbicides, or other marker nucleic acids, e.g. those involved in the development of cancer, in autoimmune diseases, degenerative diseases, infections, etc.
• the detection of amplification products may be indicative of the presence of genes conferring microbicide resistance to the captured nucleic acids of interest.
• NGS is even more specific as it provides specific sequence information of amplification products. This permits the detection and quantification of certain genes at the nucleotide level. For example, for MRSA several strains are known.
• NGS NGS
• the methods of the present invention therefore advantageously present a huge benefit vis-a-vis methods known in the art. Most of these methods are limited by the small amount of material for subsequent analysis as mentioned above. For example, usually a 5-10 ml blood sample from a human patient is used to identify and analyse potentially dangerous bacteria. Microorganisms may not be present in sufficiently large quantities. Therefore, it is presently necessary to further culture the microorganisms suspected to be present in a blood sample using culture methods.
• the enriched and captured nucleic acids of interest can be analysed using the above-described methods, and in particular molecular genetic methods, such as PCR.
• the inventive methods shorten the duration of the methods for detection of sepsis-inducing nucleic acids of interest (or any other target) and make the analysis of potentially existing microbicide (e.g. antibiotic) resistances possible in a much shorter time period, e.g.
• the fast and correct identification of nucleic acids of interest (potentially) causing sepsis in a host organism, e.g. a human being, and/or the detection of potentially existing microbicide resistances, e.g. antibiotic resistances, are of utmost importance, because these methods will permit the fast and efficient treatment of the affected individual or the prevention of disease.
• the herein disclosed methods permit a fast and accurate diagnosis and a personalized and efficient treatment of affected individuals at an earlier point in time than previous methods.
• an in vitro method according to any the preceding embodiments is provided, wherein said method is followed by a steps of selecting a suitable treatment protocol of a patient suffering from a disease, disorder or medical condition, depending on the identity, characteristics and/or quantity of the target optionally using further laboratory or clinical parameters, and optionally comprising administering a suitable treatment in terms of therapeutic agents and their concentrations, and optionally further therapeutic processes to such patient, wherein the treatment may be selected from the group comprising treatment with antibiotics, antifungal drugs, antiviral drugs, hormones, growth factors, anti-inflammatory drugs, immune serum, immunoglobulin preparations, monoclonal or polyclonal antibodies, medicaments for the stabilization of the cardiovascular system, medicaments for the treatment of hypertonia, medicaments for the treatment of hypotonia, anti-cancer drugs, and/or blood cell preparations.
• a method of monitoring the presence and/or quantity of a specific nucleic acid of interest over time and/or at different loci, e.g. loci of the organism comprising using the sampling device as defined in any of the foregoing embodiments and using a method as defined above, further comprising determining the quantity of a target at a first point in time (t°) and at least one or more points in time (t 1 to t n ), and/or comprising determining the quantity of a target nucleic acid of interest at a first site (loc°) and at least one or more sites (loc 1 to loc n ), wherein said target is selected from the group of pathogens (particularly those inducing sepsis), microorganisms, cells derived from the individual subjected to said method.
• the presence microbicide resistances as disclosed above are comprised by these methods.
• Embodiments of the invention relate to the methods for the production of device of the invention, particularly to the sampling probe comprising at least on fibre. Such methods comprise the steps:
• Embodiments of the invention relate also to a kit comprising the sampling device or probe as defined in any of the preceding claims, optionally further comprising instruction manuals for use of said sampling device and/or washing buffers, and/or devices required for post-enrichment analysis of bound target comprising chemicals selected from the group comprising antibodies and/or nucleic acids specific for a target, and/or devices for optical detection, analysis and/or measurement.
• the kits also comprise chemicals (e.g. chemical ingredients for PCR and related methods) for the detection of microbicide resistances.
• Embodiments of the invention relate to fibres, e.g. polymer fibres that are not coated with noble metals on which capture molecules are immobilized without supporting noble metal layer.
• Embodiments of the invention relate to (optical) fibres, e.g. PIR fibres that are coated with noble metals (e.g. gold), and/or to provide (optical) fibres on which capture molecules are immobilized without supporting noble metal layer.
• noble metals e.g. gold
• the fibres that can be introduced into the body without danger or with substantially reduced likelihood that the fibre breaks while inside the body.
• the detection, identification and/or analysis of nucleic acids of interest can find application in the diagnosis of medical indications, diseases or disorders of interest, in particular in the diagnosis of sepsis, the detection of sepsis-inducing pathogens prior to occurrence of symptoms of a sepsis, the characterization of sepsis-inducing pathogens in vitro, including the detection of potentially antibiotic resistant pathogens.
• Correct diagnosis allows the treating physician to select appropriate treatments, e.g. treatments with broad- spectrum antibiotic drugs or rather highly specific antibacterial drugs, for example in the treatment of bacterial strains that are resistant to one or more antibiotics, in sepsis patients.
• Embodiments of the invention as explained above relate to means and methods for the enrichment of nucleic acids of interest at the surface of a sampling probe fibre to facilitate their rapid (online) detection and to enable further detailed analysis offline after retracting the device from the body of the individual.
• the detection of the presence of microbicide resistances as disclosed above are comprised by these methods.
• the devices described herein are reusable devices comprising a sampling probe that can easily be sterilized and modified so that it allows the detection, identification, enrichment and/or analysis of nucleic acids of interest in a subsequent application.
• a sampling probe according to the invention in the detection of a first nucleic acid of interest in a first patient, and subsequently remove, sterilize by conventional methods, and modify the same as needed in order to reuse the same in the detection of the same nucleic acid or a different nucleic acid of interest in a second patient.
• the device comprises a sampling probe that comprises a silver halide fibre comprising at least one surface area decorated or conjugated with capture elements, especially antibodies, nucleic acids, or aptamers.
• the sampling probe comprises a single silver halide fibre.
• silver halide fibres e.g. PIR fibres
• PIR fibres may be covered with noble metals such as gold.
• Gold- coated PIR fibres have not been described previously. The gold layer renders the PIR fibres inert when used in vivo.
• capture elements as used herein may be immobilized directly on PIR fibres with additional noble metal, e.g., gold- layer as carrier substance.
• additional noble metal e.g., gold- layer as carrier substance.
• the latter fact substantially reduces the production costs for the herein described sampling probes.
• it is easy to remove capture molecules from the gold-coated or uncoated PIR fibres which is a big advantage as appropriately treated (e.g. disinfected) fibres may be re-used for coating with, e.g. noble metals, capture molecules and then be used in the herein disclosed methods.
• the present invention also relates to the use of the device for in situ monitoring of the presence or concentration of specific molecules, particles or microorganisms, such as e.g.
• sampling probes disclosed below can be used in the enrichment and subsequent optical analysis as well as in methods in vitro wherein enriched target are further analyzed using appropriate laboratory methods, e.g. biochemical, optical, molecular biological methods.
• the sampling device comprises a probe comprising at least one (optical) fibre, but it may also comprise at least two (optical fibres) fibres.
• individual fibres are coated with, i.e. metals, colloidal metals, metal nanoparticles, graphene, and/or some coated areas may be decorated with capture elements.
• the device comprises a light source.
• the sampling device comprises a probe, wherein said probe is suitable for insertion into the body of an individual.
• the invention further provides methods of producing the sampling devices and probes of the present invention, said method comprising:
• the device comprises a sampling probe that comprises a polymer fibre, a fibre made of fiberglass-enforced plastic, or a Plexiglas-fibre.
• a sampling probe that comprises a polymer fibre, a fibre made of fiberglass-enforced plastic, or a Plexiglas-fibre.
• polymer-coated glass fibres without additional layer of noble metals, such as gold, can be coated with a functional layer to which capture molecules can be attached.
• Respective fibres have the advantage that the material is flexible, essentially unbreakable, non-toxic, bio- and hemo compatible, i.e. that it can be safely used in vivo and that it can easily be introduced into the body via central line placements.
• coating with noble metals is also possible.
• the present invention also relates to the use of the device comprising a sampling probe that comprises a polymer fibre, a fibre made of fiberglass-enforced plastic, or a Plexiglas-fibre for in situ monitoring of the presence or concentration of specific molecules, particles or microorganisms, such as e.g. microorganisms inducing sepsis in a patient body, and/or molecules derived thereof.
• a sampling probe that comprises a polymer fibre, a fibre made of fiberglass-enforced plastic, or a Plexiglas-fibre for in situ monitoring of the presence or concentration of specific molecules, particles or microorganisms, such as e.g. microorganisms inducing sepsis in a patient body, and/or molecules derived thereof.
• sampling devices are further characterized in that they comprise a probe that is suitable for collecting, enriching, and/or analyzing nucleic acids of interest through capture elements present at its surface that selectively and specifically bind the target.
• the sampling device comprises a probe comprising at least one plastic fibre, but it may also comprise at least two plastic fibres.
• the sampling device comprises a probe comprising at least one polymer-coated glass fibre, a fibre made of fiberglass-enforced plastic, or a Plexiglas-fibre, but it may also comprise at least two such fibres. These fibres can be made of the same material, or from different materials, and may carry the same or different capture molecules.
• the silver halide fibre suitable for optical measurements comprises AgCl and/or AgBr.
• the silver halide fibre is a PIR fibre.
• the sampling device according to the present invention comprises a PIR fibre.
• the sampling device comprises a probe, which is an optical fibre.
• the sampling device comprises a probe comprising at least one silver halide fibre and at least one plastic fibre, which may be referred to as "mixed fibre".
• mixed fibres of the invention comprise at least two different fibres selected from glass-fibres, PMMA- fibres, or other fibre types that may or may not be used for optical measurements.
• the fibres have different diameters.
• the number and diameter of fibres in a mixed fibre is limited by the diameter or size of the site or cavity of an individual into which the probe shall be inserted (e.g. a vein, heart, peritoneal cavity, etc.).
• the site is a large vein
• the number of fibres and/or their diameter of a probe may be higher/larger than the diameter of a probe that is suitable for introduction in smaller veins or body cavities.
• fibre bundle In embodiments of the invention where more than one fibre is used in the sampling device, one could also refer to respective arrangements as "fibre bundle". However, the term "fibre” as used herein refers also to fibre bundles or the above mentioned “mixed fibres”. This refers also to polymer-coated glass fibres.
• the individual fibres arranged in a fibre bundle are decorated with different capture elements, so that the entirety of nucleic acids of interest bound or measured by the fibre bundle is greater or equal to the number or nucleic acids of interest bound or measured with any individual fibre.
• Some capture elements may be attached to more than a single individual fibre, for example to 1, 2, 3 or more.
• at least two fibres of identical or even different type are fused or glued together at one or more points, with or without optical leakage between the fibres.
• At least two fibres of identical or even different type comprise at least one optical element at one or both tips to divert light from one fibre to the other.
• Said optical elements comprise, i.e. U-shaped elements, elliptical or circular cones, pyramids, or prisms.
• optical elements between fibres contain gaps so that light passing from one fibre to another travels through the gap.
• the gap may be in fluid communication with the environment and may contain, i.e. gas, air, blood or other body fluids, enabling optical measurements in transmission.
• the gap may be limited by parallel or wedged surfaces, i.e. resembling rectangular or U- or V-shaped groves. The distance of the surfaces limiting the gap may also vary in any other direction, i.e. in a direction perpendicular to the optical axis of one of the fibres.
• the surfaces of the gap may be structured; and may contain, i.e. gratings, DOE, Fresnel lenses or lens like structures. Further elements may be positioned inside the gap, for example a transparent ball.
• At least one sharp element protruding from the tip or the surface of a fibre is rounded.
• the fibre is combined with other elements, e.g. plastic or metal wires, fibres, or rods, some of which may be decorated with capture elements. Wires may also be prepared to conduct electrical current, or fibres or rods may be equipped with at least one wire or conductive area attached to the fibre surface capable of conducting electrical current.
• other elements e.g. plastic or metal wires, fibres, or rods, some of which may be decorated with capture elements. Wires may also be prepared to conduct electrical current, or fibres or rods may be equipped with at least one wire or conductive area attached to the fibre surface capable of conducting electrical current.
• the diameter of the fibre is ⁇ 1 mm, or alternatively, it is larger than 1 mm, e.g. 1 mm to 3 mm, preferably, 1 to 2.5 mm. Further, it is clear that the value ⁇ 1 mm comprises any suitable fibre diameter allowing for the herein described methods, e.g. 0.01-0.99 mm.
• specific capture elements having a least one specific binding activity for a target nucleic acid of interest are disposed on the surface of the fibre. It is noted that more than one specific capture element with the same specificity or more than one capture element can be disposed on the fibre surface.
• the arrangement of the capture elements on the surface is not limited, i.e. it may be evenly distributed over the entire fibre surface or disposed only in specific regions.
• different capture element are arranged in defined ring-shaped (circular) or semi-ring shaped regions of the fibre, which preferably do not overlap, to reduce background during analysis of the bound nucleic acids of interest.
• the capture molecules are deposited in circular or rectangular regions that are spatially separated. However, any other form or shape of the binding region(s) of the capture elements is contemplated.
• the combination of fibres and/or other elements is assembled from fibres and elements thin enough so that the entire bundle is flexible, i.e. the diameter of the bundle does not exceed 2.5 mm or 1 mm.
• the fibres are combined with rods or wires or with sheathes which may be moved relatively and mostly parallel to the optical axis of the fibre, or which may stay at a fixed position with respect to the exterior while the fibre may be moved relatively to the wire, rod, or sheath.
• Such relative movement may be associated with active guidance of the device, or with exposing elements or surfaces protected by, e.g. a protective cap or cover or sheath, to the surrounding media, i.e. liquid.
• the sampling device comprises at least two or more different specific capture elements, which may be referred to as a 1 , a 2 ,...a 11 and which are disposed on the surface of the fibre, wherein said at least two of more different specific capture elements have different specificities for different target structures t 1 , t 2 ,... t n .
• the sampling probe comprises corresponding different capture elements.
• antibodies may specifically detect LPS and/or lipoteichoic acid to thereby detect an infection with gram- negative and gram-positive bacteria, respectively.
• specific capture elements directed to certain bacterial species and/or fungal/viral species may be deposited on a probe, e.g. capture elements binding specifically streptococci, staphylococci, et cetera.
• the capture elements are selected from proteins, nucleic acids, small molecules, antibodies, aptamers, and so forth. It is also possible to deposit molecules derived from bacteria, viruses, fungi or (auto-)immunogens to enrich antibodies circulating in the patient. The detection and enrichment of antibodies would allow determining whether a patient has been (recently) exposed to a certain immunogen, antigen, pathogen, or not. For example, when the capture elements are molecules derived from specific bacteria, it would be possible to find out if the patient has raised antibodies against such bacteria. Generally, the detection, enrichment and analysis of antibodies in a patient using the sampling device according to the present invention are useful in the process of monitoring the immune response over time.
• nucleic acids of interest e.g. hormones (for example in the monitoring of the patient during in vitro fertilization procedures), LPS-binding protein (LBP) as an indicator of the presence of gram-negative bacteria and/or the efficiency of a given antibiotic treatment, inflammatory markers such as C-reactive protein, TNF, GM-CSF, IL-33, IL-6, IL-12, (pro-)calcitonin, etc., or nucleic acids of interest for autoantibodies, for example myelin, et cetera.
• nucleic acids of interest e.g. hormones (for example in the monitoring of the patient during in vitro fertilization procedures), LPS-binding protein (LBP) as an indicator of the presence of gram-negative bacteria and/or the efficiency of a given antibiotic treatment, inflammatory markers such as C-reactive protein, TNF, GM-CSF, IL-33, IL-6, IL-12, (pro-)calcitonin, etc.
• the invention further provides methods of detecting, enriching, and/or analyzing a target using the sampling device and/or probe as defined in any of the foregoing paragraphs. In some embodiments, these methods are performed in vivo, in other embodiments these methods are performed in vitro.
• Nucleic acids of interest can be amplified, the quantity can be determined, or cloning steps to produce more material for subsequent genetic analyses be performed. Alternatively, it is possible to subject the molecules after removal to immune assays, fluorometric, colorimetric, radioactive, or physico-chemical tests that permit the analysis of nucleic acids of interest. Alternatively, the target molecules are not removed from the probe, but are analyzed using assays whilst they are bound to the probe surface. For example, specific binding molecules (e.g.
• antibodies or nucleic acids) carrying fluorescent moieties may be added to the bound nucleic acids of interest and after washing away unbound or non- specifically bound fluorescently-labeled binding molecules, the probe (fibre) itself may be analysed in vitro with respect to the signal-intensity (e.g. using appropriate fluorescence detecting devices). It is further possible to apply mass spectroscopic methods, e.g. MALDI or LC-MS for direct identification of nucleic acids of interest, as well as SPR.
• mass spectroscopic methods e.g. MALDI or LC-MS for direct identification of nucleic acids of interest, as well as SPR.
• the invention further provides methods of detecting, enriching, and/or analyzing a target nucleic acid using the sampling device and/or probe as defined in any of the foregoing paragraphs, which enable the establishment of a clinical diagnosis, e.g. a diagnosis of a certain disease or condition, such as a sepsis in a patient.
• a clinical diagnosis e.g. a diagnosis of a certain disease or condition, such as a sepsis in a patient.
• Respective diagnostic methods may rely on the detection of nucleic acids of interest, e.g. those of bacterial cells.
• the diagnostic methods disclosed herein may be supplemented by steps wherein bacterial cells bound to capture molecules are transferred to a suitable growth medium after removal of the probe from the patient.
• the diagnostic methods can take into account the analysis of grown bacteria, which can be further characterized using methods selected from microbiological methods, molecular genetic methods and/or physico-chemical methods.
• the present invention contemplates also combinations of methods using the herein described sampling device with other methods such as any type of PCR, including qPCR and RT-PCR or qPCR subsequent to PCR, Next Generation Sequencing, and others conventionally used techniques in clinical diagnostics.
• in vitro methods wherein obtained specifically enriched nucleic acids of interest bound to a probe as defined herein are analyzed, optionally preceded by at least one washing step in a suitable medium that ensures that a high quantity of specific nucleic acids of interest remain bound to the sampling probe while non-specifically bound material is washed away as much as possible.
• a suitable medium that ensures that a high quantity of specific nucleic acids of interest remain bound to the sampling probe while non-specifically bound material is washed away as much as possible.
• methods of selecting a suitable treatment of a patient comprising performing any of the methods referred to in the preceding sections and further comprising selecting a suitable treatment for such patient.
• Methods for the selection of a suitable treatment comprise deciding which form of treatment of a patient is suitable, e.g. a treatment with broad-spectrum or specific antibiotics, a treatment of the infected tissue (e.g. by surgery), methods involving stabilizing the metabolic functions in a patient, administering appropriate pharmaceuticals, e.g. antiinflammatory drugs, chemotherapy, administering drugs to normalize the blood pressure, the heart rate, and so forth.
• the suitable treatment is used for the treatment of patients having a sepsis, or patients at risk of developing a sepsis, e.g. those carrying a substantial amount of sepsis inducing pathogens or molecules.
• the treatment may be curative or preventive.
• it can be advisable to monitor whether or not the target is still present during or after treatment.
• the invention relates to a method of selecting a suitable treatment protocol of a patient suffering from a disease, disorder or medical condition caused by the presence or absence of a selected target, and selecting a suitable treatment for such patient depending on the identity, characteristics and/or quantity of the target optionally using further measurement data, and optionally comprising administering a suitable treatment in terms of therapeutic agents and their concentrations, and optionally further therapeutic processes to such patient.
• Such methods comprise, e.g., determining whether a microorganism (bacteria, fungi, etc.) is resistant or susceptible to certain drugs, e.g.
• antibiotics for example through establishment of an antibiogram
• MRSA antibiotic- resistant bacteria
• Klebsiella and other microorganisms referred to e.g. in the claims
• certain drugs e.g. anti-cancer drugs
• certain EGFR- antagonists such as the therapeutic antibody cetuximab that is used inter alia in the treatment of colorectal cancer, is not effective in individuals carrying certain mutations in the KRAS gene.
• Such methods will help finding a personalized treatment in an individual in need thereof.
• the methods described herein may assist in the monitoring (that is surveillance of the presence or absence of certain events over time, e.g. days, weeks, months, or years) of the development and/or occurrence of certain mutations, e.g. in microorganisms or in cancer cells.
• the device of the present invention comprising fibres decorated with capture elements as probe provides determining the presence or absence and of the quantity of nucleic acids of interest (molecules, particles or microorganisms) in a patient body without risking that the mechanical stress exerted on the fibre leads to dangerous ablation of device elements in the body.
• the diameter of the fibre is equal to or smaller than 1 mm, and preferably smaller than 500, 200, 125 ⁇ .
• the length of the fibre is between 1cm and 150 cm, e.g. between 5cm and 80cm, or between 5cm and 30cm. Further, depending on the body region that is analyzed, the length may vary. For example, a fibre comprising a probe region that is directed to an adult patient's heart may be longer than a fibre that is designed for introduction an adult patient's or even neonatal patient's arm vein.
• At least one surface area of the fibre is decorated with capture elements. Areas decorated may comprise the distal tip of the fibre, where the core of the fibre is usually exposed at the surface, or areas covering at least partially those regions of the sidewall of the fibre core where the cladding is removed.
• the cladding may be removed at the distal end of the fibre adjacent to its tip, so that in this area the entire core lies bare.
• the cladding may also be removed partially at this area at one side, so that its cross section is D-shaped with the core exposed at the flat part of the "D".
• the cross section may also have the shape of a circle missing a sector, so that it looks like a groove, for example U or V groove, parallel to the fibre.
• the groove or D shaped regions may be of limited length, for example 10 or 3 or 1 mm, and several grooves or D shaped regions may be located on the fibre, for example sequentially.
• the area partially decorated may also comprise one or several ring-like cladding regions not directly adjacent to the distal fibre tip.
• the cladding may include hole-like regions removed, which holes are partially decorated with capture elements.
• the regions where the cladding is removed may have any kind of geometrical shape dependent on the technology used to remove the cladding. While mechanical removal has shape restrictions, masked etching or laser ablation allow for an almost free choice of shape.
• the fibre may also be a core only fibre, in which case the core is partially covered by capture elements on at least one area. Fibres may be single mode or multimode fibres. Any of these fibres may be equipped with a jacket or coating at regions not decorated with capture elements.
• optical fibres light may be diverted from the core to the cladding or from the cladding to the core close to the measurement site so that the desired interaction is possible even without any removal of the cladding.
• excitation light is coupled into the fibre at an almost critical angle so that it travels to the distal tip in a "cladding mode".
• further functional elements for example crystals, conic mirrors, or diffractive optical elements, signal light is travelling back in the core, as described in EP2224270 (Al).
• the device may also comprise particular elements at the distal fibre tip.
• the tip may be covered with a dielectric mirror reflecting only selected wavelength regions of the entire spectra, for example excitation wavelengths or emission wavelengths, or wavelengths around absorption maxima.
• the mirror may also be metallic reflecting wider wavelength ranges.
• the elements may also comprise protruding or retracted cones, in particular protruding cones with a 90° opening angle or retracting ones with 90" or more, to reflect light leaving the fibre back into the fibre, or concentric gratings like Fresnel lenses to modify the acceptance angle for light leaving or entering through the tip.
• the elements may also be decorated with capture elements at the outer surface.
• the bulk of the fibre may also comprise elements influencing the light propagation within the fibre or its interaction with the surface.
• Fibre Bragg Gratings may be created in the fibre using, e.g., ultraviolet light.
• FBGs Fibre Bragg Gratings
• Several kinds of FBG are known for optical fibres as state of the art, for example to reflect selected wavelength ranges, or to divert light from the core into selected areas of the cladding or the other way round from the cladding to the core.
• the device may contain a light source at the proximal end of the fibre aligned in a way that the light emitted from the source couples to a large extent into the core or cladding of the fibre.
• optical elements may be present such as spectral filters, in particular dielectric filters, lenses including Fresnel or gradient index lenses, digital optical holograms or other micro- or nanostructures shaping the beam in wavelength dependent manner, partially transmitting mirrors, or beam splitters, i.e.
• dielectric mirrors or filters mounted at an angle of 45 degrees with respect to the fibre axis so that part of the light is reflected at an angle of 90° while the remaining part of the beam is propagating parallel to the fibre axis at the proximal tip.
• Another element may comprise a mirror placed at 45° showing a hole drilled parallel to the fibre and thus at 45° to the mirror axis, allowing collimated or focused excitation light to pass through the hole and couple into the fibre, whereas the divergent emission light leaving the proximal tip of the fibre is reflected by and large by the mirror to the detector.
• Such element may also consist of a dielectric mirror reflecting or transmitting only in a particular area where to which the excitation light is directed.
• Another element contains a grating or prism or a combination thereof to induce spectral dispersion so that a linear detector array or a matrix array may be used.
• Another element may consist of a Michelson interferometer to enable Fourier Transform Infrared Spectroscopy (FTIR).
• FTIR Fourier Transform Infrared Spectroscopy
• a light source coupled to the device may include a coherent light source comprising laser diodes or lasers, in particular quantum cascade lasers, C02 lasers, etc.
• Light sources may also comprise incoherent light sources such as LEDs or superluminescence diodes.
• Light sources may be combined to cover larger wavelength ranges, and may be pulsed or polarized. Surprisingly it was found that despite the fact that silver halide fibres were developed as Mid-IR transmitting fibres, excitation wavelength ranges from 600nm to 18 ⁇ can well be used in the device.
• Detectors may comprise photodiodes, avalanche photo diodes, charge coupled devices, charge injection devices, CMOS devices, MCT (Mercury Cadmium Telluride) or photomultipliers, other semiconductor devices like PbSe, Germanium, Gallium Arsenide. Several of them are available as line or matrix sensors, so that spectral signals can be measured in parallel.
• the device hosting the fibre may comprise a sheath made of biocompatible and especially hemocompatible material such as polyurethane, which can be inserted into a vein of the patient.
• the sheath may allow for an axial movement of the fibre induced at the proximal end of the fibre.
• the distal end of the sheath has an opening allowing blood or serum to contact the fibre surface, especially at the regions decorated with capture elements.
• the distal tip of the sheath may consist of different material attached to the sheath, for example metals.
• the distal tip may further be formed as a cap or grid so that the sharp distal tip of the fibre cannot protrude outside and insure any tissue of the patient.
• the device may be combined with other endoscopic devices, such as imaging fibre bundles, scalpels or forceps into a single unit.
• Capture elements may be directly attached to the surface of the fibre or via a linker. Coatings comprising hydrogels or sol-gels may be used to increase the surface of the decorated areas. Capture elements may also be attached to polymers like polyethylene glycol to increase the surface area.
• capture elements may be specific to particular bacteria, in particular bacteria causing sepsis, or be less specific to capture several types of bacteria, for example all gram negative or all gram positive bacteria.
• the elements may capture LPS, a structural element of the cell wall of almost all gram negative bacteria.
• Capture elements may also be directed against products secreted by bacteria, such as virulence factors, such as, for example aureolysin from staphylococcus aureus.
• Capture elements may also bind to physiological molecules of the patient to monitor levels of such molecules, for example, inflammation markers involved in Systemic Inflammatory Response Syndrome (SIRS) like interleukins 1 and 6 or Tumor Necrosis Factor alpha (TNF ).
• SIRS Systemic Inflammatory Response Syndrome
• TNF Tumor Necrosis Factor alpha
• Capture elements may also be selected to bind medication or metabolites of medication for monitoring, for example, levels of antibiotics present in the patient blood.
• the methods of collecting, enriching, detecting and analyzing nucleic acids of interest not only concerns nucleic acids of interest derived from cells foreign to the patient who is subjected to such method, but may also be derived from the patient itself (e.g. any cytokine, hormone, etc. produced by the patient in response to a stimulus, such as an infection, cancer, autoimmune disease, degenerative disorder, and so forth).
• the device may be used for optical measurement concerning molecules or cells captured, and several optical measurement methods may be employed in parallel or sequentially.
• optical measurement methods may be employed in parallel or sequentially.
• the device may include elements for measuring surface plasmon resonance comprising an excitation light source and a region of the cladding coated with metal, for example gold or platinum, but also graphene, and means for coupling the excitation light into the cladding, for example by coupling off axis with respect to the fibre as described in EP 2 224 270 Al or long periodic gratings (LPG).
• the metal coated area is located on a region of the fibre core since in this case no LPG is required, and coupling off axis is preferred, but not required.
• Such device layout is only possible due to the elastic properties of Silver halide fibres.
• Side wall coatings may be combined with a metal coating of the tip or other mirror elements inducing reflection of the excitation light in the distal fibre tip, so that the coated area is interacting twice with the excitation light.
• excitation light coupled into the fibre interacts with the metal or grapheme coating by resonating with surface plasmons, so that excitation wavelengths exciting surface plasmons are absorbed.
• the metal coating may be decorated with capture elements. When ligands like molecules or cells bind to the capture elements, the refractive index at the surface of the coating changes, inducing a plasmon resonance wavelength shift, which results in a mere change of absorbance if only one wavelength is monitored.
• the device contains regions coated with different metals or coated with a metal of different thickness so that the regions have different plasmon resonance wavelengths even if a single excitation wavelength is used for all regions.
• excitation light sources can be integrated into the device, including LEDs Laser Diodes and quantum cascade lasers, C0 2 lasers alone, and all other lasers emitting between the UV and mid IR range of the optical spectrum.
• excitation light sources may be combined to excite with a wider range of the optical spectrum.
• the excitation of several wavelength ranges may be coupled in parallel (at once) into the fibre or sequentially or in frequently repeated intermitting intervals.
• the device includes elements to measure FTIR (Fourier Transform Infrared Spectroscopy) or other implementations of Raman Spectroscopy.
• an excitation light source is required, for example a laser or laser diode, and a detector, for example CMOS, CCD or CID devices, or photodiode arrays. Both are coupled to the fibre at the proximal tip by means of a beamsplitter, and an additional mirror is mounted in a fashion movable along its optical axis, a setup which is known as Michelson interferometer. As these movements have to be performed with submicrometer precision, usually piezoelectric devices are used.
• the excitation light is passing through the cladding or bare core of the fibre, so that it is reflected back and forth between the surfaces of fibres with a diameter large compared to the wavelength of the excitation light, or it gives rise to an evanescent wave in thinner fibres.
• Raman spectroscopy uses the fact that elastic scattering of light with molecules induces a small wavelength shift. The change in wavelength depends on the molecule and the scattering electron, so that a scatter spectrum characteristic for the molecule is generated. As the excitation light is several orders of magnitudes more intense than the scattered light, it must be carefully separated, which is the reason for using the Michelson interferometer.
• Molecules present in the sample close to the fibre surface especially those close to the distal fibre tip scatter excitation light and couple it back into the fibre. But the process is only efficient for molecules attached to the fibre our bound to capture elements. Overlapping spectra from different molecule types can be separated arithmetically by fingerprinting or machine learning methods in combination with data bases for compounds and their spectra.
• Raman Spectroscopy is Surface Enhanced Raman Spectroscopy (SERS), which can also be employed with the device according to the present invention.
• SERS Surface Enhanced Raman Spectroscopy
• metallic particles with a diameter of 10 - 100 nm, for example colloidal gold are decorated with capture elements and attached to the fibre surface in regions where excitation light reaches the surface.
• the surface enhanced Raman signal is altered.
• the advantage of this method is that the Raman signal is stronger than the conventional Raman signal by several orders of magnitude.
• fluorescence quenching is used to detect cells or molecules specifically.
• excitation light is coupled into the fibre and guided to regions of the cladding, the core or the tip which are decorated with capture elements.
• the capture elements are in close vicinity to fluorescent molecules or particles, for example by conjugation or physisorption.
• the excitation light induces emission of light of longer wavelength in the fluorescent molecules or particles.
• Molecules or cells binding to the capture elements interact with the fluorescent molecules or particles, which results in a reduced emission of fluorescent light. Hence, at the wavelength of the fluorescence emission, and attenuation of the fluorescence signal is observed.
• An even higher sensitivity can be obtained using a light source which is pulsed with nanosecond or even shorter bursts, or oscillating at a high frequency, so that the fluorescence lifetime can be observed as well by attenuation of the decay curve or by a phase delay.
• Bio luminescence or Chemiluminescence is used for detection of cells or molecules.
• Luminescence may be present intrinsically in cells or molecules of interest, or may be induced by enzymes and/or luminescent substrates attached to the fibre surfaces.
• Luminescence and ELISA methods of detection may also be used for in vitro quantification of cells or molecules of interest, when enzymes and substrates are added to a test solution.
• the detection methods can be combined in the device.
• mirrors, beam splitters or fibre couplers excitation light from different sources can be coupled into the fibre.
• regions coated with metal and others uncoated regions can be designed so that they are specific for a detection method.
• Cameras and 2d matrices, linear arrays and individual point detectors can also be combined via mirrors, beam splitters or fibre couplers at the proximal tip of the fibre.
• only one light source is employed for some or even all detection methods, while spectral filters, prisms or gratings separate wavelengths of the emission spectra.
• spectral separation is combined with array detectors.
• different spectral ranges of excitation light are coupled sequentially into the fibre.
• This sequential excitation can be achieved, for example, by illuminating the proximal fibre tip with light from different sources which are sequentially switched on and off, or by inclusion of a wavelength selective element such as a scanning grating or prism in the light path between the light sources and the proximal tip.
• tuneable light sources or lasers are used for creating the excitation light, such as certain laser diodes, fibre lasers or MOP As, but also quantum cascade lasers in the infrared part of the spectrum. Their emission wavelength can be scanned over a range of wavelengths.
• capture elements are attached to regions of the fibre surface.
• Several methods are known to the expert to achieve said attachment of capture elements to conventional glass fibres, but for silver halide fibres no method has ever reported.
• capture elements are attached employing a layer-by- layer electrostatic self-assembly (ESA) technology (Arregui et al 2010).
• ESA electrostatic self-assembly
• an area of net positive or negative charge is generated on the fibre surface, for example by using plasma technology. Alternating contact of these areas with solutions of polyanions and polycations will lead to the formation of a growing thin film on top of the charged area, with alternating layers of polycations and polyanions. It is understood that other interaction forces can be used alternatively, for example hydrophilic and hydrophobic interactions.
• the contact between fibre surface areas and solution is usually made by dipping the fibre tip into the solution.
• the area coated can be limited, for example, by only exposing the areas of the fibre to the plasma discharge. This can be achieved by employing masks or by temporarily coating the areas not to be coated with a removable protective substance before plasma treatment, or a photosensitive coating which, after partly being exposed to light, is removed only at areas which were not exposed to the light.
• the protective substance can be removed after the plasma treatment or even after the entire coating procedure, depending on the properties of the protective substance.
• An example for ESA build up comprises layers of polyallylamine hydrochloride and polyacrylic acid suitable for pH sensing (Corres et al. 2007).
• the protective substance or the coating solutions are printed onto the fibre surface.
• Printing employing ink jet devices or nozzle based devices enables for direct structured coatings without requiring masks. Using printers a total absence of coating can be ensured for areas outside the intended areas.
• the coating can be structured or microstructured to achieve advantageous optical, chemical, biochemical, physical, including mechanical properties of the coating.
• Useful structures include gratings, holes, dimples, protrusions of cylindrical, triangular, rectangular or other shape. Structures can be obtained by using masks, photoresist, or printing.
• the solutions used for coating may contain capture elements or molecules or particles providing bonds (bonding elements) amenable for conjugation. It may be preferred to include bonding elements or capture elements in solutions used for all coating steps, or in just one sort of solution, or in a limited number of coating steps, in particular during the last coating step.
• capture elements are attached to areas of the fibre surface by covalent conjugation.
• a similar method was reported for functionalizing Zink ions in the ZnS surface of quantum dot nanoparticles (Chan et al. 1998).
• Silver ions at the fibre surface can be functionalized with a mercapto group by using mercaptoacetic acid, preferentially in chloroform, in particular using glacial chloroform.
• the free carboxyl group can be used for conjugating capture elements, in particular bio molecules such as antibodies, peptides or oligonucleic acids covalently.
• thiol containing ligand molecules may be attached to the silver ions.
• Suitable methods for functionalization include silanization to coat the fibre with a thin silica layer, by using for example aminoalkylsilanes or mercaptoalkylsilanes in, for example, a reverse microemulsion process.
• the amino groups of the surface may be reacted with a polymer derivative containing an N-hydroxysuccinimidyl (NHS) ester functionality to form an amide bond.
• Other useful reactions for this second step comprise Michael addition, reacting the amides with epoxides, carboxylic groups, or isocyanate derivatives, "click chemistry" reactions between azides and alkynes, and metathesis reactions.
• Phosphine oxide or thiol modified polyethylene glycol may also be used to functionalize fibres.
• surfactants for example quaternary ammonium salts in which four hydrocarbon chains are bound to a nitrogen atom that is thus positively charged, and where the counter ions are chloride or bromide associating with the silver ions of the fibre surface.
• Nanoparticle size metal preferable noble metal and preferably gold islands on the surface.
• Gold nanoparticles can be obtained, for example, by citrate reduction (Sperling et al 2010).
• the citrates may be replaced by ligands binding stronger to the particle surface, for example sulfonated phosphines or mercaptocarboxylic acids.
• Phosphines again, may be replaced by thiol- containing ligand molecules, for example thiol modified DNA as would be the case for aptamers, thiol modified peptides, or dendrimers.
• Gold nanoparticles may be precipitated on the fibre surface during any ligand replacement stage.
• Useful nanoparticles do not need to be spherical. For example, they may look rod- or disk-shaped.
• optical properties of gold nanoparticles decorated fibre differ significantly from those of bare fibres, they are, for example, perfectly suited for SERS and SPR, while ATR or Raman spectroscopy or direct fluorescence detection may suffer from the presence of gold nanoparticles.
• Capture elements on all types of fibres may further comprise fluorescent dyes or particles, so that any interaction of molecules with capture elements results in a change of the fluorescence properties, in particular in fluorescence quenching.
• the device according to the invention further comprises means to avoid or reduce binding of blood constituents, for example white or red blood cells, platelets, or proteins in regions not decorated with capture elements.
• hemocompatible coating may comprise particles or molecular layers or filaments, for example polyelectrolytes, polyacrylate, poly ethylengly col (PEG) or even proteinaceous material like serum albumin.
• the coating may be generated including a masking step, for example to separate capture and non-binding regions, and may further comprise microstructures.
• Parts of the fibre may also be coated with a plastic sheet, which itself may be coated with a hemocompatible layer.
• the device according to the invention may further be combined with other catheter compatible devices, e.g., further endoscopic optical devices like imaging fibres, fibre microscopes, or spectrometers like u-shaped evanescent wave Raman spectrometers (Almond et al. 2014).
• the device may also contain transducers close to the distal end which are connected with the proximal end of the device by means of a cable.
• the device may further comprise means for monitoring the localization of the distal end, for example by a transducer emitting electromagnetic radiation, especially in the range of radio frequency or even infrared or red wavelength domains, so that receiving transducers placed outside the body receive such signal.
• the device may be combined with a drug-eluting device, said drug-eluting device comprising a reservoir close to the distal tip of the fibre, or comprising a tube ending close to the distal end of the fibre connected to the proximal end of the fibre, where the drug is inserted, for example by means of a perfusion pump.
• the device according to the invention may further be guided into a blood vessel by inserting the fibre in a tube or intravenous catheter or cannula reducing the risk of blood vessel damage during insertion or retraction of the fibre.
• the devices according to the invention comprises the silver halide fibre combined with further elements, for example a light source, in particular a Light Emitting Diode, a Laser Diode, or a laser, detector means, and beam splitter, mirror or filter, and a numerical processer, in particular microprocessor or computer.
• the device or device combination may further be contained in a kit or set, comprising, for example, intravenous tubes, cannulas, or catheter, injection needles software controlling the device or displaying the results on a screen or sending the results, device status and other information recorded by means of cable or wireless network to other data processing devices, operating manual in paper or electronic format. It may be preferred to combine reusable elements in one set and consumables and potential reagents in a different kit.
• Inventive embodiments relate to a method to capture nucleic acids from blood using the device according to the invention, wherein said method is characterized by inserting the device into a blood vessel of a patient and keeping it in said blood vessel for a time period, for example 5, 10, 30, or 60 minutes or longer as needed, measuring at least during some part of said placement time period at least one blood parameter by an optical measurement mode of the device.
• the invention relates also to the device for use in diagnostic methods of measuring at least one blood parameter.
• Such uses also comprise in vitro analyses using obtained enriched nucleic acids of interest.
• the signal of the blood parameter is captured repeatedly or continuously during some part of said placement time period, and evaluated online or with a delay shorter than the placement time period, e.g., shorter than 1 , 10, or 30 seconds.
• This version of the method comprises also comparing the evaluated signal with a threshold or comparing a derived value, for example the difference between values measured at different time points, the derivative of the evaluated signal, or kinetic parameters calculated from a series of measured and evaluated signals, with a reference value using an electronic device, i.e. numerical processor. Once the reference value is reached, the fibre of the device is retracted automatically or a warning signal is given to the person operating the device to retract the fibre from the blood vessel.
• the signal used is a signal corresponding to the specific binding of e.g. bacteria to capture elements on the fibre surface. It may be preferred to combine at least two signals by means of a mathematical formula in a numerical processor and comparing the combined signal with a threshold value.
• the method according to the invention may include further steps as described in example No. 1.
• Example 1 Preparation and use of the sampling device
• the device according to the invention is prepared, i.e. light sources and receivers are coupled, a test signal is generated with a reference sample outside the patient body confirming the function of the capture elements fixed close to the distal fibre end, the light sources, detectors, and computers controlling the measurement devices and calculated the intended test parameters. Additional elements, such as intravenous catheters or cannulae, are prepared as well. Before starting the diagnostic procedure the sepsis suspected patient may receive other therapeutic intervention or be analyzed more generally, for example in trying to stabilize his cardiovascular system immediately without detailed diagnosis. The diagnostic procedure starts by inserting an intravenous cannula or catheter into an arm, leg, or other blood vessel, preferably vein of the patient.
• said device is inserted into the blood stream of the vessel, sometimes far enough to enter into a different blood vessel upstream or downstream from the insertion locus of the device, for example close to the heart valves to monitor potential bacterial colonization of the heart.
• a navigation system may help the operator, i.e. surgeon, to position the device precisely at the point of interest.
• the fibre portion of the device may be partly retracted to get into direct contact with the blood stream.
• the blood stream transporting bacteria of interest passes the fibre, to which the bacteria or other bacterial components bind at the regions decorated with capture elements.
• Light emitted from the light source travels through the fibre and various optical elements to regions where the capture elements are attached to the fibre surface, and gets into contact with said capture elements, augmenting or diminishing a base signal measured in absence of any medication.
• the signal often light at a different wavelength, is captured by sensor elements and the induced electrical signal is then passed to the computer, where it is numerically processed by means of cluster determination, normalization, kinetics calculation, etc.
• the positioning of the fibre in particular vessels is chosen according to the presumed or known locus of infection, in particular downstream of said locus with respect to the blood stream.
• Preferred loci comprise arm and leg veins, (cardiac catheters), or veins downstream of locations of an injury, surgery, or implants, for example stents, drug releasing implants, organ or bone or joint replacement, comprising grafted donor or artificial organs. It may further be preferred to position more than one fibre at different loci, to track different loci, or to measure differences between two loci, or to determine the quantity of target(s) across at least two loci in parallel.
• the point in time when the fibre should be retracted from the blood stream may be predetermined or sought to be calculated with computers getting online data from the device.
• the data analyzed numerically or the calculated signal derived thereof continuously or repeatedly during the presence of the fibre in the blood vessel are compared with a threshold value. Once the threshold value is exceeded, an alert is launched for the operator or physician.
• the alert signal may also be logged in computer. Furthermore, a match of the results with data base elements or lists to classify the result obtained.
• the online signal is used to control therapy parameters automatically.
• Information on specific nucleic acids of interest e.g. which derived from a pathogen, e.g. bacteria can be used to control the rate of medication released into the patient body.
• the actual perfusion speed of a perfusor can be reduced if the concentration of the antibiotic reaches or passes a threshold level, or could be increased if the antibiotic concentration is too low or the bacterial concentration increased again after treatment onset.
• the release rate of the medication may be directly or inversely proportional to the concentration signal calculated. It may be preferred to modify the calculated signal values or the threshold values according to external parameters, for example to have different values over time, in particular day or night.
• control is exerted over the state of a release valve, which could be binary, e.g., open or closed, or gradually be opened or closed according to the type of valve used. In other embodiments, control could be exerted over release mechanisms of an implant.
• a release valve which could be binary, e.g., open or closed, or gradually be opened or closed according to the type of valve used. In other embodiments, control could be exerted over release mechanisms of an implant.
• the fibre may be stripped for elements captured, so that the measured elements could be analyzed for further detail. All elements may be pooled or sampled individually. Analysis methods for said elements comprise qPCR, nanostring assays or next generation sequencing for bacteria, fungi, viruses or free oligonucleotides present in the bloodstream, Elisa, Luminex ® or similar biochemical assays, mass spectroscopy or 2d protein gel electrophoresis, or any kind of microscopy for cellular material. These tests can be performed individually or in combination to determine species or subspecies of the cells, but also antibiotic resistance. The latter can be traced, for example, by identifying specific DNA elements, in particular plasmids, specific proteins, or by Raman spectroscopy.
• the stripped cells could even be further cultured, for example as blood culture, before being further analyzed, for example to test for antibiotic resistance (i.e. to provide an antibiogram).
• optical methods are the only method class enabling such a two-step approach. All data or results or derived signals are stored on a local or network or wireless electronic storage device, and the physicians or medics are alerted by an electronic signal, sent via network or wireless to mobile devices. In addition, a local monitor or computer in vicinity of the patient bed receives similar data, which can be inspected by medical staff.
• Example 2 Enrichment of miRNA 122-sequences in an artificial blood circulation system or in "dipping"-experiments
• miRNA 122 An artificial blood circulation system comprising blood from blood reserves and externally added synthetic miDNA 122 sequences at concentrations of 10 nM or 1 nM was set to 37°C.
• the miRNAs (synthesized in form of DNA are referred to as "miDNA 122") were solubilized in 15 ml of blood and circulated in the system without external pressure or gas supplies at a velocity of 15ml/min.
• Enrichment probes were inserted for one hour into artificial circulation system.
• One region of the enrichment probe was coated with antisense DNA capture molecules that are complementary to miDNA 122.
• a different region of the enrichment probe was coated with control capture probes, namely DNA molecules that are complementary to miRNA 210 serving as negative controls.
• a further negative control region of the enrichment probe remained blank and did not carry any capture molecules.
• the enrichment probe was washed three times with PBS and bound, i.e. hybridized, targets from the respective probe areas were warmed up for 10 minutes at 95°C in 500 ⁇ of ddH 2 0 in Eppendorf tubes to elute bound target nucleic acids.
• RNA originally present in the eluted samples in ddH 2 0 was determined.
• the standard curve was obtained by amplification under the same conditions of 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM and 100 nM of synthetic miDNA 122, respectively. All samples, including those for the provision of the standard curve were subjected to qPCR in 96 well plates using a qTower (Analytik Jena). The results are shown in Table 1.
• Regions of enrichment probes spotted with specific miRNA 122 capturing molecules bound substantially more miDNA 122 than control areas. Compared with areas that do not carry any capture molecules substantially more miDNA was bound. Compared with areas carrying control DNA capture molecules consisting of non-homologous sequences (i.e. miDNA 210), those areas spotted with specific capture molecules bound 6 to 10 fault more targets (data not shown in the table).
• An artificial blood circulation system and tubes containing human blood derived from blood conserves or phosphate buffer containing synthetic DNA sequences corresponding to miRNA 122 at concentrations of 10 nM were prepared. The fluids were warmed up to 37°C. In the "dipping-experiments" tubes containing 3 ml blood or phosphate buffered saline inserted in a thermoblock set at 37°C at 300 rpm were used.
• One enrichment probe had a region that was either left blank (negative control) or that was spotted with DNA capture molecules complementary to miRNA 122.
• a second enrichment probe had a region that was either spotted with non-homologous control DNA sequence (complementary to miRNA 210) or left blank served as additional negative control.
• the probes were inserted into the fluids for one hour. Subsequent to said incubation, the bound targets were isolated and a quantitative Stem-Loop PCR was performed as described above. The results are shown in Table 2. Table 2
• miRNA spotted probes are capable of enriching specifically synthetic miDNA oligonucleotide sequences from a solution. Again it was shown that the binding efficiency is higher in blood than in the phosphate buffer system.
• Phosphate buffer in tubes comprising synthetic miRNA 122 molecules at concentrations of 1 ⁇ and 100 nM, respectively, were warmed up at a temperature of 37°C. Probes as described in Example 3 were inserted into the tube for one hour. Subsequent to the incubation, the bound molecules were eluted and a quantitative Stem-Loop PCR was performed. The amount of isolated miRNA was subsequently calculated. This example shows that also the enrichment and analysis of synthetic RNA target molecules is possible using the probes described above. The results are shown in Table 3.
• Enrichment probes carrying capture molecules were inserted for one hour into 40 ml of an E. coli (K12 strain transformed with Green Fluorescent Protein, GFP) suspension. After removal, the probes were washed three times for 10 seconds in 40 ml PBS and transferred into lysis buffer. A Promega kit and protocol StammPure Yield Plasmid Miniprep System" was used for DNA extraction. The amount of E. coli was determined by qPCR with specific primers detecting GFP.
• the enrichment probes were scratched over an agar plate for cultivation of enriched E. coli directly after removal of the enrichment probe from the animal. Results of the growth of colonies after 12 hours cultivation are shown in Figure 7.
• the left side of the petri dish (labeled "Probe 1") was scratched with an enrichment probe that was inserted for 15 min into a rat that was not inoculated with E. coli and the right side (labeled "Probe 2) shows colonies formed after scratching the agar with an enrichment probe inserted into a rat previously inoculated with E. coli.
• Example 6 Enrichment of Staphylococcus, aureus (S. aureus) in "dipping experiments” and in an artificial blood circulation system
• S. aureus were diluted at a final concentration of 10 6 colony forming units (CFU) per ml either in PBS or human blood from blood reserves.
• Enrichment probes were functionalized with anti-S. aureus antibody (ABIN341918 obtained via Antikorper-online).
• the bifunctional crosslinker Dithiobissuccimmidylpropionate (DSP) was used for the functionalization of gold surfaces on the aluminium glass fibre enrichment probes.
• Enrichment probes were inserted for one hour into 45 ml of an S. aureus suspension or inserted for 30 minutes into an artificial blood circulation system with a flow rate of 20 ml per minute using a permanent venous catheter. After removal, the probes were washed three times for 10 seconds in 40 ml PBS.
• a device for the enrichment, detection, identification and/or analysis of at least one target in vivo and in vitro characterized in that it comprises a sampling probe comprising capture elements specific for said target.
• optical signals can be measured using a method selected from the group comprising Surface Plasmon Resonance methods, RAMAN spectroscopy, Infrared Spectroscopy, fluorescence-based methods, ATR, ELISA.
• sampling device wherein at least two or more different specific capture elements are disposed on the surface of the fibre at different regions. 10. The sampling device according to any of the preceding embodiments, wherein at least two or more different specific capture elements are disposed on the surface of at least two or more different fibres, optionally at different regions of said two or more different fibres.
• the capture elements are selected from the group comprising proteins, polypeptides, antibodies, nucleic acids small molecules, aptamers, small molecules, glycolipids, lipopolysaccharides.
• sampling device comprising a light source.
• said probe is suitable for insertion into the body of an individual, wherein said individual is selected from the group comprising:
• target may further be selected from the group comprising bacteria, fungi, viruses, parasites, and toxins;
• a target of interest selected from the group comprising specific bacterial cells or fragments thereof, fungal cells or fragments thereof, virus particles or fragments thereof, parasites or fragments thereof, toxins, nucleic acids, antibodies, drugs comprising anti-cancer drugs and antibiotics;
• the sampling device according to any of the preceding embodiments, wherein the specific capture elements selectively bind bacterial cells capable of inducing sepsis in an individual or molecules derived from such bacterial cells.
• the sampling device according to embodiment 15, wherein the capture elements selectively bind bacteria selected from the group of Gram-positive and/or Gram-negative bacteria.
• the sampling device according to any of embodiments 15 and 16, wherein the capture elements selectively bind bacteria selected from the group comprising staphyloccoci including Methicillin Resistant Staphylococcus Aureus (MRS A), streptococci, gram negative gastrointestinal bacteria, E.
• MRS A Methicillin Resistant Staphylococcus Aureus
• coli Klebsiella, Enterobacter, Proteus, Pseudomonas aeruginosa, Bacteroides, or Menigococci, Haemophilus influenzae, Clostridiae, Listeriae, Salmonellae, Pasteurella multocida, Gonococci, Aeromonas, Campylobacter, Serratia marcescens, coagulase-negative Salmonellae, Acinetobacter species, Pseudomonas species, and Bacillus cereus.
• a method of detecting and/or analyzing a target indicating the presence or absence of a disease, disorder or medical indication, or a target the presence or absence of which is implicated in the development of such disease, disorder or medical condition or a target indicating the presence or absence of a therapeutic agent, in particular therapeutic molecule or its metabolites or degradation products comprising using the sampling device according to any of the preceding claims, further comprising quantifying the specific target, and optionally comparing the quantity with a threshold value.
• the method according to embodiment 18 comprising enriching specific nucleic acids of interest.
• the method according to any one of embodiments 18 and 19, wherein the specific nucleic acids of interest are bacterial cells or molecules derived from such bacterial cells.
• nucleic acids of interest are bacterial cells selected from bacteria causing sepsis, optionally selected from the bacteria referred to in claims 16 and 17.
• An in vitro method for the detection, analysis and/or quantification of specific nucleic acids of interest wherein (i) the cell type and/or (ii) the molecular target structure and/or (iii) the quantity of obtained nucleic acids of interest bound to a probe as defined in any one of embodiments 1 to 17 are determined, optionally preceded by at least one washing step, further optionally comprising comparing the results of said detection, analysis and/or quantification method with threshold value(s) or reference value(s).
• nucleic acids of interest are characterized in combination with at least one method selected from the group comprising:
• microbiological methods optionally comprising preparing an antibiogram and/or the identification of nucleic acids of interests,
• molecular biologic methods comprising methods for the characterization of the identity and/or quantity and/or mutational status of nucleic acids and/or polypeptides and/or glycoproteins or glyco lipids,
• (f) 2d protein gel electrophoresis A method of selecting a suitable treatment protocol of a patient suffering from a disease, disorder or medical condition caused by the presence or absence of a selected target, comprising performing any of the methods referred to in embodiments 18 to 25, and selecting a suitable treatment for such patient depending on the identity, characteristics and/or quantity of the target optionally using further measurement data, and optionally comprising administering a suitable treatment in terms of therapeutic agents and their concentrations, and optionally further therapeutic processes to such patient.
• the treatment is selected from the group comprising treatment with antibiotics, antifungal drugs, antiviral drugs, hormones, growth factors, anti-inflammatory drugs, immune serum, immunoglobulin preparations, monoclonal or polyclonal antibodies, medicaments for the stabilization of the cardiovascular system, medicaments for the treatment of hypertonia, medicaments for the treatment of hypotonia, anti-cancer drugs, and/or blood cell preparations.
• a method of monitoring the quantity of a specific target over time and/or at different loci comprising using the sampling device according to any one of claims 1 to 17 in a method according to any one of embodiments 18 to 27, further comprising determining the quantity of a target at a first point in time (t°) and at least one or more points in time (t 1 to t n ), and/or comprising determining the quantity of a target at a first site (loc°) and at least one or more sites (loc 1 to loc n ).
• said target is selected from the group of pathogens, microorganisms, cells derived from the individual subjected to said method, molecules derived from pathogens, microorganisms or the individual, and/or molecules that have been administered to said individual optionally selected from the group comprising medicaments optionally further selected from anti-cancer drugs comprising antibodies against cancer-specific antigens, antibiotics and antiviral drugs .
• a kit comprising the sampling device or probe as defined in any one embodiments 1 to 17, optionally further comprising instruction manuals for use of said sampling device and/or washing buffers, and/or devices required for post-enrichment analysis of bound target comprising chemicals selected from the group comprising antibodies and/or nucleic acids specific for a target, and/or devices for optical detection, analysis and/or measurement.
• a device for capturing, enrichment, detection, identification and/or analysis of a nucleic acid particularly characterized in that it comprises a sampling probe comprising capture elements specific for said nucleic acid.
• the probe is suitable for use in vivo in a subject, wherein said subject is selected from the group of subjects suspected to be at risk of development of a sepsis, subjects suspected to be infected by a microorganism, particularly microorganisms that have developed resistance to a microbicide, subjects suspected or at risk of harboring a cancer cell, subjects that are or may be pregnant.
• the probe comprises a glass fibre or does not comprise a glass fibre, optionally comprising a fully polymeric fibre.
• the glass fibre comprises a polymer coating, preferably comprising at least one polymer selected from the group comprising hydrogels, polyethylene (PE), polypropylene (PP), polyethyleneterephtalat (PET), polyvinylchloride (PVC), polycarbonate (PC), polystyrene, polyamine, polyamide (PA), polytetraf uoroethylene (PTFE), polymethylmethacrylate (PMMA), polyurethane (PUR), polysiloxane, polyether ketone (PEEK), polysulfone (PSU), polyhydroxyethylmethacrylate (PHEMA), polyimide (PI) or combinations thereof, or wherein the fully polymeric fibre comprises at least one polymer selected from the group comprising hydrogels, polyethylene (PE), polypropylene (PP), polyethyleneterephtalat (PET), polyvinylchloride (PVC), polycarbonate (PC), polystyrene,
• a target attached to a capture molecule is analyzed using a method selected from the group comprising mass spectrometry methods, Surface Plasmon Resonance methods, RAMAN spectroscopy, Infrared Spectroscopy, fluorescence-based methods, FISH, ATR, ELISA, molecular biologic methods, in particular (RT-)PCR, and NGS.
• sampling device according to any one of embodiments 31 to 35, wherein the diameter of the fibre is ⁇ _1 mm, ⁇ 0.9 mm, ⁇ 0.8 mm, ⁇ 0.7 mm, ⁇ 0.6 mm, or ⁇ 0.5 mm.
• the sampling device according to any one of embodiments 31 to 36, wherein specific capture elements having specific for at least one nucleic acid of interest are disposed on the surface of the probe.
• the sampling device according to any of embodiments 31 to 37, wherein at least two or more different specific capture elements a 1 , a 2 ,...a n are disposed on the surface of the fibre, wherein said at least two of more different specific capture elements have different specificities for different nucleic acids of interest NAI 1 , NAI 2 ,...
• NAI n The sampling device according to any of embodiments 31 to 38, wherein at least two or more different specific capture elements are disposed on the surface of the fibre at different regions.
• the sampling device according to any of embodiments 31 to 40, wherein the capture elements are selected from the group comprising antibodies, nucleic acids including PNA, small molecules, aptamers, random oligonucleotides, peptides Ca+ ions, Cs- ions, which are chelating agents binding targets due to ionic interactions.
• sampling device according to any of embodiments 31 to 41, wherein the nucleic acid of interest is bound via hydrogen bonding, ionic interactions, particularly through ion chelator complexes and silicates.
• said probe is suitable for insertion into the body of an individual, wherein said individual is selected from the group comprising:
• the sampling device according to any of embodiments 31 to 43, wherein the specific capture elements selectively bind nucleic acids derived from bacterial cells capable of inducing sepsis in an individual or molecules derived from such bacterial cells.
• the sampling device according to embodiments 43, wherein the capture elements selectively bind nucleic acids derived from bacteria selected from the group comprising staphyloccoci including Methicillin Resistant Staphylococcus Aureus (MRSA), streptococci, gram negative gastrointestinal bacteria, E.
• MRSA Methicillin Resistant Staphylococcus Aureus
• coli Klebsiella, Enterobacter, Proteus, Pseudomonas aeruginosa, Bacteroides, or Menigococci, Haemophilus influenzae, Clostridiae, Listeriae, Salmonellae, Pasteurella multocida, Gonococci, Aeromonas, Campylobacter, Serratia marcescens, coagulase-negative Salmonellae, Acinetobacter species, Pseudomonas species, and Bacillus cereus.
• a method of enriching, detecting and/or analyzing a nucleic acid indicating the presence or absence of a disease, disorder or medical indication, or a nucleic acid the presence or absence of which is implicated in the development of such disease, disorder or medical condition comprising using the sampling device according to any of embodiments 31 to 44, further comprising qualitatively and/or quantitatively analyzing the specific target, and optionally comparing the quantity with a threshold value.
• the method according to embodiments 46 or 47 comprising enriching specific nucleic acids, particularly those that are associated with the presence of or the development of infection, of genetic disorders, of autoimmune diseases, of metabolic disorders, of sepsis or cancer. .
• targets are characterized in combination with at least one method selected from the group comprising:
• Mass spectroscopic methods comprising MALDI-ToF or LC-MS.
• a method of selecting a suitable treatment protocol of a patient suffering from a disease, disorder or medical condition caused by the presence or absence of a source comprising or releasing a nucleic acid of interest comprising performing any of the methods referred to embodiments 46 to 54, and selecting a suitable treatment for such patient depending on the identity, characteristics and/or quantity of the nucleic acid of interest, optionally using further measurement data, and optionally comprising administering a suitable treatment in terms of therapeutic agents and their concentrations, optionally wherein said patient suffers from sepsis.
• a method of stratifying a patient for a suitable treatment protocol comprising performing any of the methods referred to in claims embodiments 46 to 54, and selecting a suitable treatment for such patient depending on the identity, characteristics and/or quantity of the nucleic acid of interest, optionally using further measurement data, and optionally comprising administering a suitable treatment in terms of therapeutic agents and their concentrations, optionally wherein said patient suffers from sepsis.
• the treatment is selected from the group comprising treatment with antibiotics, antifungal drugs, antiviral drugs, hormones, growth factors, anti-inflammatory drugs, immune serum, immunoglobulin preparations, monoclonal or polyclonal antibodies, medicaments for the stabilization of the cardiovascular system, medicaments for the treatment of hypertonia, medicaments for the treatment of hypotonia, anti-cancer drugs, drugs for the treatment of autoimmune diseases, and/or blood cell preparations.
• the treatment is selected from the group comprising treatment with antibiotics, antifungal drugs, antiviral drugs, anti-inflammatory drugs, immune serum, immunoglobulin preparations, monoclonal or polyclonal antibodies, medicaments for the stabilization of the cardiovascular system, medicaments for the treatment of hypertonia, medicaments for the treatment of hypotonia, and/or blood cell preparations.
• a method of monitoring the quantity of a nucleic acid of interest over time and/or at different loci comprising using the sampling device according to any one of claims 1 to 14 in a method according to any one of claims 46 to 58, further comprising determining the quantity of a nucleic acid of interest at a first point in time (t°) and at least one or more points in time (t 1 to t n ), and/or comprising determining the quantity of a nucleic acid of interest at a first site (loc°) and at least one or more sites (loc 1 to loc n ).
• nucleic acid is derived from any one comprised by the group of pathogens, microorganisms, and cells derived from the individual, particularly cancer cells, or cancer cells subjected to a therapeutic treatment method.
• the method according to embodiment 61, wherein the steps of analyzing the nucleic acids for presence and/or absence of resistances to at least one microbicide compound, particularly at least one antibiotic comprise the amplification of nucleic acid molecules associated with microbicide resistance and detection and optionally further analysis of the amplification product.
• a kit comprising the sampling device or probe as defined in any one embodiments 31 to 44, optionally further comprising instruction manuals for use of said sampling device and/or washing buffers, and/or devices required for post-enrichment analysis of bound nucleic acids comprising chemicals selected from the group comprising antibodies and/or nucleic acids specific for a target, and/or devices for optical detection, analysis and/or measurement.

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