EP3152570A1

EP3152570A1 - Means and methods for the detection of targets

Info

Publication number: EP3152570A1
Application number: EP16724398.9A
Authority: EP
Inventors: Joachim DYCK; Jens Lenk; Stefan Sauermann
Original assignee: Yaya Diagnostics GmbH
Current assignee: Yaya Diagnostics GmbH
Priority date: 2015-08-06
Filing date: 2016-05-19
Publication date: 2017-04-12
Also published as: WO2017021023A1

Abstract

The present invention relates to means for and methods of collecting, enriching, and analyzing targets of interest, which are associated with the development of sepsis. The means may be used in vivo or in vitro using obtained targets. The invention can, inter alia, be used in the detection and enrichment as well as analysis of target cells such as pathogens or molecules derived thereof or from individuals containing such pathogens, wherein these pathogens of molecules derived thereof are involved in the development of sepsis.

Description

Means and methods for the detection of targets

Field of the invention

The present invention relates to devices and methods for the enrichment of targets, particularly cells, such as microorganisms, and/or cell-derived or microorganism- derived molecules, in particular infectious, very particularly sepsis-inducing microorganisms and/or molecules thereof in the diagnosis of infections or sepsis, for the identification of sepsis-inducing cells or molecules and detection and determination of antibiotic susceptibility or resistance in the causative agent capable of inducing sepsis. In particular, the present invention relates to a device comprising a sampling probe that selectively binds biological targets and/or molecules as well as to methods involving the use of such device. Background of the invention

The analysis of body fluids is a main task of human diagnostics, particularly in the diagnosis of infectious agents or molecules derived therefrom. Usually, a body fluid sample comprising, for example, blood, saliva, spinal fluid or urine is collected and brought into contact with 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. Alternatively, 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.

Further, due to the increasing knowledge of pathogenic or endogenous cells or molecules in blood, new approaches for better or faster diagnostic tests have emerged. 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.

In the diagnosis or detection of cancer Circulating Tumor Cells (CTC) 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. In a preferred embodiment, 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, however, delivers only off-line signals, i.e. after retraction of the steel wire from the body.

It would, however, also be desirable to provide new and improved means and methods for the detection and characterization of biological targets, e.g. cells and microorganisms or target molecules in general suspected to be present in an individual or in other environments suspected of containing them.

Presently, with the notable exception of biopsies, blood cell counts and PAP smear tests, whole cells are rarely analyzed for diagnostic purposes. One reason for this fact can be the scarcity of some cells in body fluids. For example, concentrations of circulating tumor cells are estimated at 1 cell per milliliter (ml), while white blood cells are usually present in the order of 10⁶ per ml, and red blood cells at 10⁹ per ml. By contrast, cell numbers of microorganisms such as bacteria present in blood usually range in the order of 1 - 1,000 cells per ml in samples derived from individuals suffering from infections and/or sepsis, compared with samples from healthy individuals.

Although the number of bacteria in patients suspected to suffer from sepsis is frequently elevated, the diagnosis of sepsis or the identification of the causative agent, e.g. bacteria, usually require lengthy culturing and proliferation using blood cultures, a process in which bacteria grow for several hours or days to obtain sufficient numbers of bacterial mass for further characterization, e.g. for the detection of antibiotic resistances, which are often associated with severe consequences for the patients and the health system. Similarly, fungal/yeast infections that may also cause sepsis, e.g. through Candida strains, require cultivation of the pathogen, immunoassays, etc. before a specific treatment with fungicides can be applied. Further, a large number of infectious agents are difficult to culture in cell- free media in vitro. This limits also the possibility of obtaining sufficient material for analysis, characterizing the same, e.g., to detect antibiotic resistances.

Late onset of treatment of sepsis is associated with an increased mortality risk.

Thus a rapid diagnostic test for sepsis is urgently required, in particular in view of the high risk of mortality. According to a recent publication of the National Institutes of Health more than one million patients per year are affected by severe sepsis in the United States alone (http://www.nigms.nih.gov/Education/Pages/factsheet_sepsis.aspx), whereof about 28-50 % dies. Further, immediate treatment is of particular importance in sepsis cases. The survival rate of patients treated after onset of the initial symptoms of a septic shock is 77% when the treatment starts later than 30 minutes, whereas only 42% of such patients survive when the treatment starts later than 5 hours after onset of such symptoms (Kumar et al, Crit Care Med 2006, Vol. 34, No. 6, pp. 1589-1596). In Germany, sepsis is the third most frequent death cause after cardiovascular diseases and cancer. The German Federal Ministry for Education and Research (Bundesministerium fur Bildung und Forschung, BMBF) estimates that on average about 160 individuals per day (about 60,000 per year in an German population of about 80 millions) die from sepsis in Germany, which is almost as high as the number of casualties due to heart attacks (cf. http ://www. gesundheitsforschung-bmbf. de/ dc/774.php ; 10 April 2015).

It would be desirable to measure both, off-line signals and biological parameters that could enable the tracing of variations of parameters of interest over time. Such 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 of certain target cells and/or target molecules involved in the development of sepsis, e.g. in the detection, analysis and/or quantification of pathogens such as microorganisms of molecules derived thereof. Ideally, such means and methods would preferably also comprise the identification of pathogens as well as the determination of antibiotic resistances.

In the past, efforts were made to enable online measurements using fibre optics. In respective devices, blood components interacting with the glass surface of the fibre are measured. Suitable optical methods comprise surface plasmon resonance (SPR), fluorescence, Raman and infrared spectroscopy, in particular reflectance, transmittance, and surface enhanced Raman spectroscopy (SERS). It has been suggested to combine these methods with the use of capture molecules attached to the fibre surface. Capture molecules could be directly attached to the glass surface, or to a coated region of the glass surface. For example, such device has been disclosed for tracing E. coli bacterial cells in vitro (Maraldo et al. 2006). The above physical methods may also overcome inherent problems encountered in many PCR-based tests for the presence of microorganisms, which can be particularly bothersome in detection of sepsis. In PCR assays, recombinant Taq Polymerase is used in most cases, i.e. an enzyme that is frequently produced in recombinantly modified bacteria such as E. coli. Despite serious efforts to purify the recombinantly produced enzyme from the recombinant hosts, contamination with host-derived nucleic acids is still a problem. This can affect reliable diagnosis of, e.g., infections of the urogenital system, the gastro-intestinal tract or bile ducts by enterococcae (e.g. E. coli or EHEC). Also sepsis due to E. coli may be difficult to diagnose by PCR. Therefore, improved means and methods are urgently required. The present invention addresses this need.

Moreover, an important disadvantage of previous approaches relying on the insertion of a sampling device into the body of an individual, said device comprising a probe in form of a fibre is the small surface area of the fibre tip. This reduces the number of different capture molecule species which can be attached to said surface. Generally, the fibre end surface is so small that only weak signals are generated. Moreover, light passing through the fibre hits the fibre tip surface almost perpendicularly, so that SPR measurements are virtually impossible. It has been suggested to strip the plastic coating from the fibre to attach capture molecules to the bare surface of the cladding. However, the light, has to be guided to these surface of the fibre by expensive means, for example by fibre Bragg gratings (FBG) or tapers. Such means increase production complexity and cost of the fibre variants. Furthermore, stripped fibre sections are very brittle, which is prohibitive for their in vivo use. For these reasons, these devices have still not found any use in routine in vivo diagnostics.

It was suggested by others to use silver halide fibres for in vivo measurements. Usually these fibres are step index fibres made of AgCl and AgBr, and the high refractive index core is due to increased Bromine to Chlorine concentration ratio in the fibre core compared to the fibre cladding. These fibres exhibit excellent mechanical flexibility and are much less fragile than conventional fibres. It is even possible to abandon plastic fibre jackets partly or totally, and without risk of releasing fibre born particles and ablated fragments into the environment. Silver halide fibres have only been used in the infrared part of the wavelength spectrum where they exhibit the highest transparency.

Mackanos et al. (2010) disclose a fibre optic spectrometer for online measurement in vivo employing silver halide fibres. Both fibres of their Y-probe are joint at the distal fibre ends by a diamond prism so that light emitted from one fibre is coupled back into the second fibre after being reflected twice under 90° at the surfaces of the diamond prism. The proximal end of the second fibre is connected to a Michelson interferometer. The interaction between biological sample and device takes place at the diamond prism surface. A second variant disclosed by the same authors employs a single fibre bent by 180° and a radius of about 4 mm. The bend induces coupling of the light travelling in the fibre with the fibre surface. The authors designed these devices with tissue analysis in mind. Due to the strong absorbance of mid IR light by water, all analysis has to take place close to the fibre surface. The authors show ex vivo and in vivo measurement results, the latter being limited, however, to measurements performed in larger and natural body orifices like mouth, bladder, or colon. The fibre device diameter of 4 mm or more is prohibitive, however, for use in arm veins.

Boettger et al. (2009) and Artyushenko (EP2224270 (Al)) have simplified this set up by replacing the Y-shaped probe by a single fibre. The distal end face of the fibre hosts a conical tip reflecting light back into the same fibre where it came from. At the proximal and of the fibre, a camera or microscope is attached. The authors suggest the use of the fibre device in blood vessels, but the device is not capable of collecting or enriching cells or other targets at the same time.

With respect to the detection of sepsis-inducing microorganisms and determination of antibiotic resistances others attempt to develop point-of-care devices for isothermal nucleic acid amplification of nucleic acids derived from patient samples as well as RAMAN-spectroscopy for the identification of sepsis-inducing germs (Schubert et al, Laborwelt 4/2011, pages 9-10), however, so far these aims were not achieved. One of the reasons therefore, in particular with respect to nucleic acid amplification, could be the lack of sample material available for a characterization of the pathogens and/or molecules inducing sepsis. For example, samples with small volumes taken from a patient may not provide a sufficient number of cells and/or molecules for further analysis using molecular diagnostic methods or spectroscopic diagnostic methods. Others, e.g., in the Iridica™ system (Abbott), have attempted to improve the detection of sepsis-inducing bacteria by means of oligonucleotide primers directed to conserved regions of various bacteria, fungi and viruses. The initial patient sample usually consists of 3 to 10 ml blood. This amount, however, frequently does not contain sufficient material to detect and analyze sepsis-inducing pathogens reliably.

Therefore, none of the known devices and/or methods solves the problem of collecting and enrichment of target cells or target molecules at the surfaces of the device in situ, e.g. whilst it is inserted in a blood vessel, and analyzing said targets either online without the risk of shedding fibre components which would inevitably lead to complications for the patient

Further, devices for the enrichment or analysis of targets, e.g. target cells or molecules derived therefrom are not known, after removal of a device from an organism in vitro, in particular of targets derived from patients (such as patients suspected to be carriers of sepsis-inducing organisms) are not known. Still further, it was not possible to enrich targets using devices to be inserted in vivo into an organism, removing the same from said organism, analyzing bound targets (e.g. identifying pathogens or molecules derived therefrom), for example by means of cell culturing methods in a suitable medium, and subsequently or in parallel performing a molecular genetic analysis of the bound material, e.g. using PCR and/or other nucleic acid analysis techniques (for example Next Generation Sequencing). Such devices and respective methods are helpful in the detection, identification and characterization of antibiotic resistances.

Summary of the invention

It is an object of the present invention to provide means, such as sampling devices comprising probes and uses thereof in the enrichment, detection, identification and/or analysis of target cells and/or target molecules capable of inducing sepsis 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 (blood, CSF), wherein the probes are suitable for the enrichment of targets in vivo or ex vivo (e.g. from blood cultures, blood reserves, urine, extracorporeal environmental sources, etc.). It is also an object that bound targets can be analyzed after removal of the probe from the blood vessel or body cavities, to analyze enriched targets in vitro, e.g. using cell culture, molecular genetic or other methods, such as MALDI or FISH (fluorescence in situ hybridization)-based methods.

Brief Description of the Figures

Fig. 1 : shows a fibre optic set up as contemplated in embodiments of the invention. In this example, a step index fibre is used with a core (103) of higher and a cladding (102) of lower refractive index. A jacket (101) protects the fibre from mechanical damage. Jacket and cladding are removed at some areas so that light travelling in the fibre interacts with the surface of the fibre. A coating (113) made of metals or other, i.e. conducting, material may cover the core at least partly to enable 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 antibodies or their derivatives, or aptamers, or other polypeptides and their derivatives 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 (1 17) to the second coating (1 15), and capture element (114) to the first coating (113). The fibre may comprise further coatings (109) in other areas of the exposed fibre surfaces, i.e. metal coating 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 elements like bacterial (112) or eukaryotic cells, fungi, endogenous or exogenous molecules, viruses (116) and/or their respective fragments. Furthermore, 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, 114, 117, 118, or 111) may comprise further, in particular optically active elements (108), i.e. fluorescent dyes. For a gradient index fibre, the cladding (102) is omitted, the remainder of the figure would be identical.

Fig. 2 shows examples of different embodiments of fibre bundles according to the invention in cross section. As in Fig. 1, the examples shown comprise only step index fibres while the same principles apply for gradient index and photonic crystal fibres in a similar way. 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 an embodiment of the invention, relating to one exemplary workflow for use of the device of the invention. Accordingly, the device is introduced into the environment suspected of containing targets, e.g. target microorganisms or target cells or target molecules (collectively called "target") and left in place for a desired period of time (e.g. 5 min or more, for 15 min or more, preferably for 30 to 60 minutes, for about 1.5 hours, for about 2 hours or more, for about 3 hours or more, for about 4 hours or more, or for about 5 hours or more, during which period of time targets binding to the device are enriched). Subsequently, in case of living target organisms, these may be inoculated on a solid growth medium (e.g. blood agar) and/or into a liquid growth medium (e.g. in a blood culture vessel), or the bound and enriched targets are directly processed for analysis, or they may be processed for analysis after cultivation on solid growth medium or in liquid growth medium. The enriched targets may be cultivated on solid media or in liquid media for a desired period of time, e.g. for 1 to 72 hours as needed, following standard cultivation procedures, e.g. for bacterial cultures. Subsequent to the cultivation step (which may be omitted in some embodiments of the invention), bound organisms can be identified (e.g. biochemically, using molecular biology methods, such as (RT-)PCR or using fluorescence-based in situ hybridization (FISH) methods, or physically, e.g. using mass spectrometry methods, optic methods, and the like). In parallel, or subsequently, the presence of antibiotic resistances can be determined, e.g. by means of an antibiogram, by way of measurement of the optical density of growth media containing defined antibiotic compounds at defined concentrations, or mass-spectrometrically, optically, etc.. Several methods can be performed consecutively or in parallel. Figure 5 shows a standard procedure for the identification of microorganisms, e.g. bacteria, suspected of causing sepsis and susceptibility testing thereof. First a liquid sample, e.g. a blood sample, is obtained from an environment suspected of containing targets (for example bacteria, particularly sepsis-inducing bacteria). The liquid sample (e.g. whole blood, blood plasma, cell cultures-derived liquid samples, etc.) suspected of containing a target is transferred to a liquid growth medium such as a blood culture vessel. The liquid culture is incubated under standard culture conditions for a period of between about 12-72 hours. Subsequently, a liquid culture sample is plated out on a solid growth medium (e.g. blood agar in a petri dish) and incubated overnight (e.g. 4-12 hours). Grown bacterial colonies are picked and transferred into a solution (e.g. NaCl, PBS, citrate buffer, etc.). Thereafter, a turbidity-based standardization of the cell count (e.g. McFarland) is performed and subsequently an identification and/or antibiotic susceptibility testing according to standard protocols known in the art. It is also possible, to perform biochemical analyses or additional antibiogram analyses in parallel or subsequent to the cell count determination. The entire protocol from the sampling to the identification of bacteria takes about 48 to 120 hours while susceptibility testing conventionally takes about 60 to 144 hours.

Figure 6 shows a workflow embodiment according to the invention for the identification of targets, here targets suspected of causing sepsis, as well as susceptibility testing thereof. First, the device of the invention is introduced for a period of about 5 min to 120 min, for example for 30-60 minutes, into an environment in vivo or ex vivo suspected of containing biological targets (such as target organisms such as microorganisms, for example bacteria, particularly sepsis-inducing bacteria), e.g. a blood vein or a blood culture vessel. The targets are enriched on/in the device surface due to their specific adherence to binding partners that are attached to the surface of the inventive device. After removal of the device from the environment that is analyzed, the bound enriched targets are inoculated on a solid growth medium (e.g. a blood agar) and the inoculated agar plate is subjected to suitable cultivation conditions, e.g. overnight (e.g. 4-12 hours) in a suitable environment allowing for the growth of the biological targets (standard procedures can be used for bacterial cultures). Colonies of organisms that are grown are picked and transferred into a solution (e.g. NaCl, PBS, citrate buffer etc.). Thereafter, a McFarland turbidity-based standardization of the cell count and subsequently an identification and/or antibiotic susceptibility testing is performed according to standard protocols known in the art. It is also possible, to perform biochemical analyses or additional antibiogram analyses in parallel or subsequent to the cell count determination. The entire protocol from the sampling to the identification of bacteria takes about 30 to 48 hours while susceptibility testing takes about 48 to 72 hours. The identification and susceptibility testing steps can be performed in parallel or consecutively.

Figure 7 shows a workflow embodiment according to the invention for the identification of targets, here targets suspected of causing sepsis and susceptibility testing thereof. First, the device of the invention is introduced for a period of about 5 min to 120 min, for example for 30-60 minutes, into an environment in vivo or ex vivo suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria, particularly sepsis-inducing bacteria), e.g. a blood vein or a blood culture vessel. The targets are enriched on/in the device surface due to their specific adherence to binding partners that are attached to the surface of the inventive device. After removal of the device from the environment that is analyzed, the bound enriched targets are inoculated into liquid growth medium (e.g. a Brain-Heart Bouillon) and subjected to cultivation conditions for about 30 minutes to 12 hours. Thereafter, in one example the enriched targets, which are have proliferate on/in the device as substrate are directly inoculated on a solid growth medium (e.g. a blood agar) from the device, and in an alternative targets, which have proliferated in the liquid growth medium, i.e. targets that have proliferated in the medium as free-floating cells, are transferred to a solid growth medium (e.g. blood agar) and in all cases the inoculated agar plate is subjected to suitable cultivation conditions, e.g. overnight (e.g. 4-12 hours) in a suitable environment allowing for the growth of the biological targets (standard procedures can be used for bacterial cultures). Colonies of organisms that are grown are picked and transferred into a solution (e.g. NaCl, PBS, citrate buffer, etc.). Thereafter, a turbidity-based standardization of the cell count (e.g. McFarland) and subsequently an identification and/or antibiotic susceptibility testing is performed according to standard protocols known in the art. It is also possible, to perform biochemical analyses or additional antibiogram analyses in parallel or subsequent to the cell count determination. The entire protocol from the sampling to the identification of bacteria takes about 24 to 60 hours while susceptibility testing takes about 48 to 84 hours. The identification and susceptibility testing steps can be performed in parallel or consecutively.

Figure 8 shows an embodiment according to the invention for the identification of targets, in this case, identification of targets suspected of causing sepsis, and susceptibility testing thereof. First, the device of the invention is introduced for a period of about 5 min to 120 min, for example for 30-60 minutes, into an environment in vivo or ex vivo suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria, particularly sepsis-inducing bacteria), e.g. a blood vein or a blood culture vessel. The targets are enriched on/in the device surface due to their specific adherence to binding partners that are attached to the surface of the inventive device. After removal of the device from the environment that is analyzed, the bound enriched targets are inoculated into liquid growth medium (e.g. a Brain-Heart Bouillon) and subjected to cultivation conditions, e.g. for about 30 minutes to 12 hours in a suitable environment to allow for growth of the targets (standard procedures can be used for bacterial cultures). A sample of the growth medium is transferred into solution (e.g. NaCl, PBS, etc.) and subjected to a turbidity-based standardization of the cell count (e.g. McFarland) and subsequently the identification and/or antibiotic susceptibility testing is performed according to standard protocols known in the art. The identification and susceptibility testing steps can be performed in parallel or consecutively. It is possible, to perform biochemical analyses or additional antibiogram analyses in parallel, subsequent to the cell count determination. The entire protocol from the sampling to the identification of bacteria takes about 24 to 48 hours while susceptibility testing takes about 48 to 72 hours.

Figure 9 shows an embodiment according to the invention for the identification of targets, in this case, identification of targets suspected of causing sepsis, and susceptibility testing thereof. First, the device of the invention is introduced for a period of about 5 min to 120 min, for example for 30-60 minutes, into an environment in vivo or ex vivo suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria, particularly sepsis-inducing bacteria), e.g. a blood vein or a blood culture vessel. The targets are enriched on/in the device surface due to their specific adherence to binding partners that are attached to the surface of the inventive device. After removal of the device from the environment that is analyzed, the bound enriched targets are inoculated into liquid growth medium (e.g. a Brain-Heart Bouillon) and subjected to cultivation conditions, e.g. for about 30 minutes to 12 hours in a suitable environment to allow for growth of the targets (standard procedures can be used for bacterial cultures). A sample of the growth medium is subjected to a turbidity-based standardization of the cell count (e.g. McFarland) and subsequently the identification and/or antibiotic susceptibility testing is performed according to standard protocols known in the art. The identification and susceptibility testing steps can be performed in parallel or consecutively. It is possible, to perform biochemical analyses or additional antibiogram analyses in parallel, subsequent to the cell count determination. The entire protocol from the sampling to the identification of bacteria takes about 24 to 48 hours while susceptibility testing takes about 48 to 72 hours.

Figure 10 shows an embodiment according to the invention for the identification of targets, here bacteria suspected of causing sepsis and susceptibility testing thereof. First, the device of the invention is introduced for a period of about 5 min to 120 min, for example 30-60 minutes, into an environment in vivo or ex vivo suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria, particularly sepsis-inducing bacteria), e.g. a blood vein or a blood culture vessel. The targets are enriched on/in the device surface due to their specific adherence to binding partners that are attached to the surface of the inventive device. After removal of the device from the environment that is analyzed, the bound enriched targets in one alternative are detached (e.g. by way of physical or chemical methods, e.g. using sonication shifting the pH value, shifting the ionic strength, applying an electrical field or cleaving enzymatically) from the device and inoculated into liquid growth medium (e.g. a Brain-Heart Bouillon) and subjected to cultivation conditions, e.g. for about 30 minutes to 12 hours in a suitable environment to allow growth of the targets (standard procedures can be used for bacterial cultures). In a second alternative, the bound targets are transferred into liquid growth medium and detached from the device (using, e.g. physical or chemical methods) which is subsequently retracted from the vessel containing the liquid growth medium, and subsequently the target organisms are incubated in liquid growth medium (e.g. for about 30 minutes to 12 hours in a suitable environment to allow growth of the targets). Thereafter, a sample of the growth medium is transferred to a solid growth medium (e.g. blood agar) and incubated for a time sufficient to form colonies (e.g. overnight (e.g. 4-12 hours)). In both alternatives, grown colonies are transferred into a solution (e.g. NaCl, PBS, citrate buffer, etc.) and subsequently subjected to a turbidity-based standardization of the cell count (e.g. McFarland) and subsequently the identification and/or antibiotic susceptibility testing is performed according to standard protocols known in the art. It is also possible, to perform biochemical analyses or additional antibiogram analyses in parallel or subsequent to the cell count determination. The identification and susceptibility testing steps can be performed in parallel or consecutively. The entire protocol from the sampling to the identification of bacteria takes about 30 to 60 hours while susceptibility testing takes about 48 to 84 hours.

Figure 11 shows an embodiment according to the invention for the identification of targets, here bacteria suspected of causing sepsis and susceptibility testing thereof. First, the device of the invention is introduced for a period of about 5 min to 120 min, for example 30-60 minutes, into an environment in vivo or ex vivo suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria, particularly sepsis-inducing bacteria), e.g. a blood vein or a blood culture vessel. The targets are enriched on/in the device surface due to their specific adherence to binding partners that are attached to the surface of the inventive device. After removal of the device from the environment that is analyzed, the bound enriched targets in one alternative are detached (e.g. by way of using physical or chemical methods, such as sonication, shifting the pH value, shifting the ionic strength, applying an electrical field or cleaving enzymatically) from the device and inoculated into liquid growth medium (e.g. a Brain-Heart Bouillon) and subjected to cultivation conditions, e.g. for about 30 minutes to 12 hours in a suitable environment to allow growth of the targets (standard procedures can be used for bacterial cultures). In a second alternative, the targets bound on/in the device are transferred into liquid growth medium and there detached from the device (using, e.g. physical or chemical methods) which is subsequently retracted from the vessel containing the liquid growth medium, and subsequently the target organisms are incubated in liquid growth medium (e.g. for about 1 to 12 hours in a suitable environment to allow growth of the targets). Thereafter, a sample of the growth medium is either transferred to a solution (e.g. NaCl, PBS, citrate buffer, etc.) and then subjected to a turbidity-based standardization of the cell count (e.g. McFarland) and subsequently an identification and/or antibiotic susceptibility testing is performed according to standard protocols known in the art, or a sample of the organisms grown in liquid growth medium is directly subjected to a turbidity-based standardization of the cell count (e.g. McFarland) and subsequently an identification and/or antibiotic susceptibility testing is performed according to standard protocols known in the art. It is also possible, to perform biochemical analyses or additional antibiogram analyses in parallel or subsequent to the cell count determination. The identification and susceptibility testing steps can be performed in parallel or consecutively. The entire protocol from the sampling to the identification of bacteria takes about 24 to 48 hours while susceptibility testing takes about 48 to 72 hours. Figure 12 shows an embodiment according to the invention for the identification of targets, here bacteria suspected of causing sepsis and susceptibility testing thereof. First, the device of the invention is introduced for a period of about 5 min to 120 min, for example for 30-60 minutes, into an environment in vivo or ex vivo suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria, particularly sepsis-inducing bacteria), e.g. a blood vein or a blood culture vessel. The targets are enriched on/in the device surface due to their specific adherence to binding partners that are attached to the surface of the inventive device. After removal of the device from the environment that is analyzed, the bound enriched targets may be detached (e.g. by way of physical or chemical methods) from the device and inoculated into solution (e.g. NaCl, PBS, citrate buffer, etc.) and subsequently subjected to a turbidity-based standardization of the cell count (e.g. McFarland) and subsequently the identification and/or antibiotic susceptibility is performed according to standard protocols known in the art. It is also possible, to perform biochemical analyses or additional antibiogram analyses in parallel or subsequent to the cell count determination. The identification and susceptibility testing steps can be performed in parallel or consecutively. The entire protocol from the sampling to the identification of bacteria takes about 24 to 36 hours while susceptibility testing takes about 48 to 60 hours.

Figure 13 shows a standard procedure using MALDI TOF MS based identification and susceptibility testing of bacteria suspected of causing sepsis. First a liquid sample, e.g. a blood sample, is obtained from an environment suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria, particularly sepsis- inducing bacteria). The liquid sample suspected of containing targets is transferred to a liquid growth medium such as a blood culture vessel. The liquid culture is incubated under standard culture conditions for a period of between about 12-72 hours. Subsequently, a liquid culture sample is plated out on a solid growth medium (e.g. blood agar in a petri dish) and incubated overnight (e.g. 4-12 hours). Grown bacterial colonies are picked (e.g. using a pipet tip) and transferred to a MALDI target (e.g. using MALDI matrix, such as a-Cyano-4- hydroxycinnamic acid (CHCA) or 2,5-dihydroxybenzoic acid (DHB)). Thereafter, the picked organisms are crystallized on the MALDI target and subjected to MALDI identification and MALDI-based antibiotic susceptibility or resistance testing. It is possible to perform a parallel turbidity-based standardization of the cell count (e.g. McFarland) and subsequently an identification and/or antibiotic susceptibility testing according to standard protocols known in the art (see Figure 5). It is also possible, to perform identification or additional antibiogram analyses in parallel or consecutively - subsequent to the cell count determination. The entire protocol from the sampling to the identification of bacteria/susceptibility testing conventionally takes about 2 to 4 days.

Figure 14 shows a workflow embodiment of the present invention using

MALDI TOF MS for the identification and susceptibility testing of targets, e.g. bacteria suspected of causing sepsis and susceptibility testing thereof. First, the device of the invention is introduced for a period of about 5 min to 120 min, for example 30-60 minutes, into an environment in vivo or ex vivo suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria, particularly sepsis-inducing bacteria,), e.g. a blood vein or a blood culture vessel. The targets are enriched on/in the device surface due to their specific adherence to binding partners that are attached to the surface of the inventive device. After removal of the device from the environment that is analyzed, the device is optionally subjected to a washing step (e.g. using physiological saline or PBS) to remove blood components that could interfere with detection, and the bound enriched targets are transferred to the MALDI target and further processed using MALDI matrix (e.g. using MALDI matrix, such as CHCA or DHB on/in the device for MALDI-TOF MS preparation). Thereafter, the organisms are crystallized on the MALDI target and subjected to MALDI TOF MS based identification and MALDI TOF MS-based antibiotic susceptibility and/or resistance testing. The entire protocol from the sampling to the identification usually takes 1-2 hours; for susceptibility testing it takes usually about 1-24 hours.

Figure 15 shows a workflow embodiment of the present invention using MALDI for the identification and susceptibility testing of targets, e.g. bacteria, suspected of causing sepsis and susceptibility testing thereof. First, the device of the invention is introduced for a period of about 5 min to 120 min, for example 30-60 minutes, into an environment in vivo or ex vivo suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria, particularly sepsis-inducing bacteria), e.g. a blood vein or a blood culture vessel. The targets are enriched on/in the device surface due to their specific adherence to binding partners that are attached to the surface of the inventive device. After removal of the device from the environment that is analyzed, the device is optionally subjected to a washing step (using e.g. physiological saline) to remove blood components that could interfere with detection. Thereafter, the device is deposited on a solid growth medium (e.g. a blood agar plate) for the purpose of inoculation. The microorganisms bound on/in the device or parts of the device proliferate and grow colonies on the solid growth medium and/or on the device itself. Targets grown on the solid growth medium (e.g. a blood agar plate) or grown on the device are incubated at suitable conditions for about 1 - 12 hours. Colonies grown on the device or on the solid growth medium are subsequently picked, e.g. using a pipette, and processed using MALDI matrix (e.g. using MALDI matrix, such as CHCA or DHB) on MALDI targets directly with MALDI-matrix on MALDI targets. In another alternative, colonies are not picked, but the device is removed from the solid growth medium and transferred to the MALDI target, where attached targets are processed using MALDI matrix (e.g. using MALDI matrix, such as CHCA or DHB. In both alternatives, the organisms are crystallized on the MALDI target and subjected to MALDI identification and MALDI- based antibiotic susceptibility testing. The entire protocol from the sampling to the identification usually takes 1 to 12 hours; for susceptibility testing it takes usually about (e.g. about 1 to 36 hours). It is possible to perform a parallel turbidity-based standardization of the cell count (e.g. McFarland) and subsequently an identification and/or antibiotic susceptibility testing according to standard protocols known in the art. It is also possible, to perform identification or additional antibiogram analyses in parallel or consecutively - subsequent to the cell count determination.

Figure 16 shows a workflow embodiment of the present invention using MALDI for the identification and susceptibility testing of targets, e.g. bacteria, suspected of causing sepsis and susceptibility testing thereof. First, the device of the invention is introduced for a period of about 5 min to 120 min, for example 30-60 minutes, into an environment in vivo or ex vivo suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria, particularly sepsis-inducing bacteria,), e.g. a blood vein or a blood culture vessel. The targets are enriched on/in the device surface due to their specific adherence to binding partners that are attached to the surface of the inventive device. After removal of the device from the environment that is analyzed, the device is optionally subjected to a washing step to remove blood components that could interfere with detection. Thereafter, the device is transferred into liquid growth medium (e.g. a Brain-Heart-Bouillon) where they are incubated under suitable conditions for about 1 to 12 hours to allow target organisms to proliferate and multiply either adherent to the device or as non-attached organisms in the liquid growth medium. After a sufficiently long incubation time in the growth medium to permit proliferation of the target organism on the device itself, said device may be transferred directly to a MALDI target (with target organisms attached) and subjected to MALDI matrix. Prior to the administration of the matrix, the fragment is optionally subjected to a washing step to remove growth medium or blood components. Further, for cases where the organisms grow in the liquid growth medium, e.g. as non-adherent targets, all or a part of the liquid growth medium is removed after a sufficiently long cultivation to harvest the grown target organisms by means of centrifugation (according to standard procedures known to a person skilled in the art, e.g. a microbiologist). After removal of the supernatant, the pelleted target organisms are optionally washed, centrifuged again, and then at least a part of the pellet is transferred to a MALDI target, e.g. using a pipette tip. Alternatively, a sample of the organisms grown in the liquid growth medium can be directly transferred, without centrifugation step, e.g. using a pipette. In all cases the transferred targets are exposed to MALDI matrix (e.g. such as a-Cyano-4-hydroxycinnamic acid (CHCA) or 2,5- dihydroxybenzoic acid (DHB)) directly on MALDI targets. The organisms are crystallized on the MALDI target and subjected to MALDI identification and MALDI-based antibiotic susceptibility testing. The entire protocol from the sampling to the MALDI TOF MS based identification and susceptibility testing usually takes about 1-24 hours; It is possible to perform a parallel turbidity-based standardization of the cell count (e.g. McFarland) from the centrifuged pellets and subsequently an identification and/or antibiotic susceptibility testing according to standard protocols known in the art. It is also possible, to perform identification or additional antibiogram analyses in parallel or consecutively - subsequent to the cell count determination.

Figure 17 shows a workflow embodiment of the present invention using MALDI TOF MS for the identification and susceptibility testing of targets, e.g. bacteria, suspected of causing sepsis and susceptibility testing thereof. First, the device of the invention is introduced for a period of about 5 min to 120 min, for example 30-60 minutes, into an environment in vivo or ex vivo suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria,, particularly sepsis-inducing bacteria,), e.g. a blood vein or a blood culture vessel. The targets are enriched on/in the device surface due to their specific adherence to binding partners that are attached to the surface of the inventive device. After removal of the device from the environment that is analyzed, the device is optionally subjected to a washing step to remove blood components that could interfere with detection. Thereafter, the device, i.e. the part of the device where targets are captured and enriched, is transferred into liquid growth medium (e.g. a Brain-Heart-Bouillon). The attached targets are detached using physical or chemical methods (e.g. using agitation, wiping off, or enzymatically or chemically detaching the bound targets from binding molecules, e.g. using enzymes, solutions having a shift in pH or ionic strength or the like) while the sampling device is in the vessel containing the growth medium and after detachment the sampling device is removed from the vessel. Alternatively, the targets are detached from the device (e.g. by way of using sonication shifting the pH value, shifting the ionic strength, applying an electrical field or cleaving enzymatically, e.g. by way of incorporating a peptidic sequence recognized by enzymes such as peptidases so that the bound material is released; the peptidase may generally in respective steps be added to the growth media or can be added separately) prior to transferring the same into the vessel containing liquid growth medium. In both alternatives, the liquid growth medium containing the inoculate is incubated under suitable conditions for about 1 to 12 hours to allow target organisms to proliferate and multiply. After a sufficiently long incubation time of the growth medium to permit proliferation of the target organism, all or a part of the medium is removed to harvest the grown target organisms by means of centrifugation (according to standard procedures known to a person skilled in the art, e.g. a microbiologist). After removal of the supernatant, the pelleted target organisms are optionally washed, centrifuged again, and then at least a part of the pellet is transferred to a MALDI target, e.g. using a pipette. Alternatively, a sample of the organisms grown in the liquid growth medium can be directly transferred e.g. using a pipette, i.e. without centrifugation step. In all cases the transferred targets are exposed to MALDI matrix (e.g. such as a-Cyano-4-hydroxycinnamic acid (CHCA) or 2,5-dihydroxybenzoic acid (DHB)) directly on MALDI targets. The organisms are crystallized on the MALDI target and subjected to MALDI identification and MALDI-based antibiotic susceptibility testing. The entire protocol from the sampling to the identification of bacteria/susceptibility testing usually takes about 1-24 hours. It is possible to perform a parallel turbidity-based standardization of the cell count (e.g. McFarland) and subsequently an identification and/or antibiotic susceptibility according to standard protocols known in the art. It is also possible, to perform biochemical analyses or additional antibiogram analyses in parallel, subsequent to the cell count determination. Figure 18 shows a workflow embodiment of the present invention using MALDI for the identification of targets, e.g. bacteria suspected of causing sepsis and susceptibility testing thereof. First, the device of the invention is introduced for a period of about 5 min to 120 min, for example 30-60 minutes, into an environment in vivo or ex vivo suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria, particularly sepsis-inducing bacteria), e.g. a blood vein or a blood culture vessel. The targets are enriched on/in the device surface due to their specific adherence to binding partners that are attached to the surface of the inventive device. After removal of the device from the environment that is analyzed, the device is optionally subjected to a washing step to remove blood components that could interfere with detection. Thereafter, the device with enriched targets, i.e. the part of the device where targets are captured and enriched, is transferred into liquid growth medium (e.g. a Brain-Heart-Bouillon). The attached targets are detached using physical or chemical methods (e.g. using agitation, wiping off, or enzymatically or chemically detaching the bound targets from binding molecules, e.g. using enzymes, or the like) while the sampling device is in the vessel containing the growth medium and after detachment the sampling device is removed from the vessel. The liquid growth medium containing the inoculate is incubated under suitable conditions for about 1 to 12 hours to allow target organisms to proliferate and multiply. After a sufficiently long incubation time of the growth medium to permit proliferation of the target organism, all or a part of the medium is removed to harvest the grown target organisms by means of centrifugation (according to standard procedures known to a person skilled in the art, e.g. a microbiologist). After removal of the supernatant, the pelleted target organisms are optionally washed, centrifuged again, and then at least a part of the pellet is transferred to a MALDI target, e.g. using a pipette. The organisms grown in the liquid growth medium can also be directly transferred e.g. using a pipette, where they are exposed to MALDI matrix (e.g. such as a-Cyano-4-hydroxycinnamic acid (CHCA) or 2,5-dihydroxybenzoic acid (DHB)) directly on MALDI targets. The organisms are crystallized on the MALDI target and subjected to MALDI identification and MALDI-based antibiotic susceptibility testing. The entire protocol from the sampling to the identification of bacteria/susceptibility testing usually takes about 1- 24 hours. It is possible to perform a parallel turbidity-based standardization of the cell count (e.g. McFarland) and subsequently an identification and/or antibiotic susceptibility according to standard protocols known in the art. It is also possible, to perform biochemical analyses or additional antibiogram analyses in parallel, subsequent to the cell count determination.

Figure 19 (a+b) show a workflow embodiment of the present invention using MALDI for the identification of targets, e.g. bacteria suspected of causing sepsis and susceptibility testing thereof. First, the device of the invention is introduced for a period of about 5 min to 120 min, for example 30-60 minutes, into an environment in vivo or ex vivo suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria, particularly sepsis-inducing bacteria), e.g. a blood vein or a blood culture vessel. The targets are enriched on/in the device surface due to their specific adherence to binding partners that are attached to the surface of the inventive device. After removal of the device from the environment that is analyzed, the device is optionally subjected to a washing step to remove blood components that could interfere with detection. Thereafter, the device is transferred into liquid growth medium (e.g. a Brain-Heart-Bouillon). The attached targets are detached using physical or chemical methods (e.g. using agitation, wiping off, or enzymatically or chemically detaching the bound targets from binding molecules, e.g. using enzymes, or the like) while the sampling device is in the vessel containing the growth medium and after detachment the sampling device is removed from the vessel. Targets detached in to the liquid growth medium are incubated under suitable conditions for about 1 to 12 hours to allow target organisms to proliferate and multiply. After a sufficiently long incubation time of the growth medium to permit proliferation of the target organism, all or a part of the medium may optionally be removed to harvest the grown target organisms by means of centrifugation (according to standard procedures known to a person skilled in the art, e.g. a microbiologist). After removal of the supernatant, the pelleted target organisms are optionally washed and centrifuged again. A part of the pellet (provided a harvesting step by centrifugation is performed at all) or a sample of the liquid growth medium containing the targets (i.e. without prior centrifugation harvesting step) is transferred to a solid growth medium (e.g. blood agar plate). Alternatively, the targets are detached from the device and transferred directly onto the solid growth medium (i.e. without prior incubation in liquid growth medium). In all cases the target organisms are incubated under suitable culturing conditions on a solid growth medium for about 1 to 12 hours to allow the formation of colonies prior to transferring the same to MALDI targets, e.g. using a pipette, and subjected them to MALDI matrix ( e.g. such as a-Cyano-4-hydroxycinnamic acid (CHCA) or 2,5-dihydroxybenzoic acid (DHB)). The organisms are crystallized on the MALDI target and subjected to MALDI-based identification and MALDI-based antibiotic susceptibility testing. The entire protocol from the sampling to the identification of bacteria/ susceptibility testing usually takes about 1-36 hours in the alternative with cultivation in liquid and on solid growth media and 1-24 hours in the alternative without incubation in liquid growth medium, respectively. It is possible to perform a parallel turbidity-based standardization of the cell count (e.g. McFarland) and subsequently an identification and/or antibiotic susceptibility according to standard protocols known in the art. It is also possible, to perform biochemical analyses or additional antibiogram analyses in parallel or subsequent to the cell count determination.

Figure 20 shows a workflow embodiment of the present invention using

MALDI for the identification of targets, e.g. bacteria suspected of causing sepsis and susceptibility testing thereof. First, the device of the invention is introduced for a period of about 5 min to 120 min, preferably for 30-60 minutes, into an environment in vivo or ex vivo suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria, particularly sepsis-inducing bacteria), e.g. a blood vein or a blood culture vessel. The targets are enriched on/in the device surface due to their specific adherence to binding partners that are attached to the surface of the inventive device. After removal of the device from the environment that is analyzed, the device is optionally subjected to a washing step to remove blood components that could interfere with detection. Thereafter, the device with enriched targets is transferred into e.g. a buffer and the targets are detached from the device ((e.g. by way of using sonication shifting the pH value, shifting the ionic strength, applying an electrical field or cleaving enzymatically) prior to transferring the same directly to MALDI target, e.g. using a pipette tip and exposed to MALDI matrix (e.g. with CHCA or DHB) with or without prior centrifugation step. The organisms are crystallized on the MALDI target and subjected to MALDI-based identification and MALDI-based antibiotic susceptibility testing. It is possible to perform both ways of MALDI-based identification of targets and susceptibility testing in parallel or consecutively, wherein either alternative may be performed prior to the second alternative. The entire protocol from the sampling to the identification of bacteria / susceptibility testing usually takes 1-4 hours.

Figure 21 shows a standard procedure using PCR-based identification of bacteria suspected of causing sepsis, and susceptibility testing thereof. First a liquid sample, e.g. a blood sample, is obtained from an environment suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria, particularly sepsis-inducing bacteria). The liquid sample suspected of containing a target is transferred to a liquid growth medium, such as a blood culture. The liquid culture is incubated under standard culture conditions for a period of between about 12-72 hours. Subsequently, in one alternative workflow, a complex and long-lasting (e.g. 30 min to 4 hours) procedure for removal of e.g. whole blood components, nucleic acids (R A and/or DNA) are isolated from organisms that have grown in liquid growth. With the term "complex and long-lasting procedure" the large number of steps that have to be performed in order to extract DNA/RNA from whole blood samples. Whole blood is quite difficult to handle due to the presence of various ingredients (cells, proteins, and the like) and due to the coagulation of the therein comprised ingredients belonging to the cascade of coagulation-factors. Further, unlike in the present invention, the weight volume ratio between the blood sample of usually only a few milliliters and the amount of nucleic acids contained therein is quite high, i.e. nucleic acids are present only in minute amounts, particularly nucleic acids of potentially rare infectious targets. The organisms may be harvested by centrifugation and optionally washing the obtained pellet of organisms after decanting the supernatant. The pelleted organisms are suspended in a suitable buffer and lysed to release nucleic acids. Nucleic acids are isolated and purified using standard methods known in the art, e.g. in a diagnostic, molecular biology or clinical microbiology laboratory. The nucleic acids are subjected to Reverse transcription when the target nucleic acid is RNA and then subjected to a specific PCR or using isothermal amplification. Alternatively, when the isolated nucleic acid target is DNA, no Reverse transcription is required and a specific PCR is performed using genomic or plasmid DNA. The (RT-)PCR conditions and reagents (in particular the primers and optionally probes) are selected to allow specific (RT-)PCR-based detection of target organisms of interest, e.g. to identify one or more sepsis-inducing microorganisms. Where the genes conferring susceptibility or resistance to selected antibiotics are known, it is contemplated to use PCR as a means to detect those genes involved and thereby determine the targets' susceptibility/resistance to selected antibiotics and the like. The entire protocol from the sampling to the identification of bacteria/susceptibility testing usually takes about 1 to 4 days. In an alternative workflow, the target organisms grown either in the liquid growth medium or, in yet a further alternative, the liquid with suspected target organisms obtained from the sample source are both subjected to semi-automated procedures for sample preparation and analysis with instruments using cartridges or bags loaded with required substances for processing positive blood culture bottles. In yet another alternative, a semi-automated method without culturing step allows for direct use of 0.5 -3.0ml of liquid sample without use positive liquid culture medium, e.g. blood culture bottle for sample preparation and analytics, e.g. DNA amplification and melting curve analysis. It is also possible to perform a parallel turbidity-based standardization of the cell count (e.g. McFarland) and subsequently an identification and/or antibiotic susceptibility according to standard protocols known in the art. It is further possible to perform biochemical analyses or additional antibiogram analyses in parallel, subsequent to the cell count determination.

Figure 22 (a+b) show an inventive workflow using PCR-based identification of targets, e.g. bacteria suspected of causing sepsis, and also susceptibility testing thereof. First, the device of the invention is introduced for a period of about 5 min to 120 min, for example 30-60 minutes, into an environment in vivo or ex vivo suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria, particularly sepsis-inducing bacteria), e.g. a blood vein or a blood culture vessel. The targets are enriched on/in the device surface due to their specific adherence to binding partners that are attached to the surface of the inventive device. After removal of the device from the environment that is analyzed, the device is optionally subjected to at least one quick washing step (e.g. 1 - 10 min) to remove components that could interfere with any of the following steps. The target organisms attached to the sampling device of the invention are inoculated on a solid growth medium, e.g. a blood agar plate. After a sufficient time (e.g. about 1 to 12 hours) at suitable culturing conditions, the grown colonies are picked from the solid growth medium and subjected to nucleic acid isolation and purification according to known methods. Briefly, targets are suspended in a suitable buffer and lysed to release nucleic acids. Nucleic acids are isolated and purified using standard methods known in the art, e.g. in a diagnostic, molecular biology or clinical microbiology laboratory. The nucleic acids are subjected to Reverse transcription when the target nucleic acid is RNA and then subjected to a specific PCR or isothermal amplification. Alternatively, when the isolated nucleic acid targets is DNA, no Reverse transcription is required and a specific PCR or isothermal amplification is performed using genomic or plasmid DNA. The (RT-)PCR conditions and reagents (in particular the primers and optionally probes) are selected to allow specific (RT-)PCR-based detection of target organisms of interest, e.g. to identify one or more sepsis-inducing microorganisms. The same applies also to workflows involving isothermal amplification of nucleic acid. When the genes conferring susceptibility or resistance to selected antibiotics are known, it is contemplated to use PCR as a means to detect those genes involved and thereby determine the targets' susceptibility/resistance to selected antibiotics and the like. The entire protocol from the sampling to the identification of bacteria/susceptibility testing usually takes about 6 hours to 4 days. Above mentioned procedures for sample preparation and analysis can be performed (semi-)automatically with instruments using cartridges or bags loaded with required substances for processing colonies grown on solid growth media. Another semi-automated method allows for direct use of 0.5-3.0ml of liquid suspension of colonies grown on solid growth media for sample preparation and analytics, e.g. DNA/R A amplification and melting curve analysis. It is also possible to perform a parallel turbidity-based standardization of the cell count (e.g. McFarland) and subsequently an identification and/or antibiotic susceptibility according to standard protocols known in the art. It is further possible to perform biochemical analyses or additional antibiogram analyses in parallel, subsequent to the cell count determination.

Figure 23 (a+b) show an inventive workflow using PCR-based identification of targets, e.g. bacteria suspected of causing sepsis, and also susceptibility testing thereof. First, the device of the invention is introduced for a period of about 5 min to 120 min, preferably for 30-60 minutes, into an environment in vivo or ex vivo suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria, particularly sepsis-inducing bacteria), e.g. a blood vein or a blood culture vessel. The targets are enriched on/in the device surface due to their specific adherence to binding partners that are attached to the surface of the inventive device. After removal of the device from the environment that is analyzed, the device is optionally subjected to at least one quick washing step to remove components that could interfere with any of the following steps. The target organisms attached to the sampling device of the invention are inoculated into a liquid growth medium, e.g. a Brain-Heart- Bouillon. After a sufficiently long incubation time of the growth medium to permit proliferation of the target organisms (e.g. about 1 to 12 hours), all or a part of the medium may optionally be removed from the culture vessel to harvest the grown target organisms by means of centrifugation (according to standard procedures known to a person skilled in the art, e.g. a microbiologist). After removal of the supernatant, the pelleted target organisms are optionally washed and centrifuged again and subjected to nucleic acid isolation and purification according to known methods. Briefly, targets are suspended in a suitable buffer and lysed to release nucleic acids. Nucleic acids are isolated and purified using standard methods known in the art, e.g. in a diagnostic, molecular biology or clinical microbiology laboratory. The nucleic acids are subjected to Reverse transcription when the target nucleic acid is RNA and then subjected to a specific PCR or isothermal amplification. Alternatively, when the isolated nucleic acid targets is DNA, no Reverse transcription is required and a specific PCR or isothermal amplification is performed using genomic or plasmid DNA. The (RT-)PCR conditions and reagents (in particular the primers and optionally probes) are selected to allow specific (RT-)PCR-based detection of target organisms of interest, e.g. to identify one or more sepsis-inducing microorganisms. The same applies also to isothermal nucleic acid amplification methods. When the genes conferring susceptibility or resistance to selected antibiotics are known, it is contemplated to use PCR as a means to detect those genes involved and thereby determine the targets' susceptibility/resistance to selected antibiotics and the like. The entire protocol from the sampling to the identification of bacteria/susceptibility testing usually takes about 6 hours to 4 days. Above mentioned procedures for sample preparation and analysis can be performed (semi-)automatically with instruments using cartridges or bags loaded with required substances for processing target organisms proliferated in said liquid growth media. Another semi-automated method allows for direct use of 0.5-3.0ml of suspension with target organisms proliferated in said liquid growth media for sample preparation and analytics, e.g. DNA/RNA amplification and melting curve analysis. It is also possible to perform a parallel turbidity-based standardization of the cell count (e.g. McFarland) and subsequently an identification and/or antibiotic susceptibility according to standard protocols known in the art. It is further possible to perform biochemical analyses or additional antibiogram analyses in parallel, subsequent to the cell count determination. The entire protocol from the sampling to the identification of bacteria/susceptibility testing usually takes about 6 to 18 hours.

Figure 24 shows an inventive workflow using PCR-based identification of targets, e.g. bacteria suspected of causing sepsis, and also susceptibility testing thereof. First, the device of the invention is introduced for a period of about 5 min to 120 min, preferably for 30-60 minutes, into an environment in vivo or ex vivo suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria, particularly sepsis-inducing bacteria), e.g. a blood vein or a blood culture vessel. The targets are enriched on/in the device surface due to their specific adherence to binding partners that are attached to the surface of the inventive device. After removal of the device from the environment that is analyzed, the device is optionally subjected to at least one quick washing step to remove components that could interfere with any of the following steps. The target organisms attached to the sampling device of the invention are transferred into a suitable buffer and lysed to release nucleic acids. Nucleic acids are isolated and purified using standard methods known in the art, e.g. in a diagnostic, molecular biology or clinical microbiology laboratory. The nucleic acids are subjected to Reverse transcription when the target nucleic acid is RNA and then subjected to a specific PCR or isothermal nucleic acid amplification. Alternatively, when the isolated nucleic acid targets is DNA, no Reverse transcription is required and a specific PCR or isothermal nucleic acid amplification is performed using genomic or plasmid DNA. The (RT-)PCR conditions and reagents (in particular the primers and optionally probes) are selected to allow specific (RT-)PCR-based detection of target organisms of interest, e.g. to identify one or more sepsis-inducing microorganisms. The same applies also to isothermal nucleic acid amplification methods. When the genes conferring susceptibility or resistance to selected antibiotics are known, it is contemplated to use PCR as a means to detect those genes involved and thereby determine the targets' susceptibility/resistance to selected antibiotics and the like. It is possible to perform quantitative measurements of numbers of target organisms bound on a defined area on device using e.g. Q-PCR since said targets are not subjected to unspecific endpoint DNA/RNA amplification or to incubation on solid or in liquid growth media. The entire protocol from the sampling to the identification of bacteria/susceptibility testing usually takes about 2 to 6 hours. Above mentioned procedures for sample preparation and analysis can be performed (semi-)automatically with instruments using cartridges or bags loaded with required substances for processing target organisms which are transferred directly from the device to suspension, e.g. PBS. Another semi- automated method allows for direct use of 0.5-3.0ml of suspension with target organisms which were transferred directly from the device to suspension, e.g. PBS for sample preparation and analytics, e.g. DNA/RNA amplification and melting curve analysis. It is also possible to perform a parallel turbidity-based standardization of the cell count (e.g. McFarland) and subsequently an identification and/or antibiotic susceptibility according to standard protocols known in the art. It is further possible to perform biochemical analyses or additional antibiogram analyses in parallel, subsequent to the cell count determination. Figure 25 (a+b) show a workflow embodiment of the present invention using (RT-)PCR for the identification of targets, e.g. bacteria suspected of causing sepsis and susceptibility testing thereof. First, the device of the invention is introduced for a period of about 5 min to 120 min, preferably for 30-60 minutes, into an environment in vivo or ex vivo suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria, particularly sepsis-inducing bacteria), e.g. a blood vein or a blood culture vessel. The targets are enriched on/in the device surface due to their specific adherence to binding partners that are attached to the surface of the inventive device. After removal of the device from the environment that is analyzed, the device is optionally subjected to at least one quick washing step to remove blood components that could interfere with detection. Thereafter, the device with enriched targets is transferred into liquid growth medium (e.g. a Brain-Heart- Bouillon). The attached targets are detached using physical or chemical methods (e.g. using agitation, wiping off, or enzymatically or chemically detaching the bound targets from binding molecules, e.g. using enzymes, solutions having a shift in pH or ionic strength or the like) while the sampling device is in the vessel containing the growth medium and after detachment the sampling device is removed from the vessel. Alternatively, the targets are detached from the device prior to transferring the same into the vessel containing liquid growth medium. The liquid growth medium is incubated under suitable conditions for about 1 to 12 hours to allow target organisms to proliferate and multiply. After a sufficiently long incubation time of the growth medium to permit proliferation of the target organism, all or a part of the medium is removed to harvest the grown target organisms by means of centrifugation (according to standard procedures known to a person skilled in the art, e.g. a microbiologist). After removal of the supernatant, the pelleted target organisms are optionally washed and centrifuged again. Alternatively, a sample of the liquid culture is directly used, i.e. without prior centrifugation. The target organisms attached to the sampling device of the invention are transferred into a suitable buffer and lysed to release nucleic acids. Nucleic acids are isolated and purified using standard methods known in the art, e.g. in a diagnostic, molecular biology or clinical microbiology laboratory. The nucleic acids are subjected to Reverse transcription when the target nucleic acid is RNA and then subjected to a specific PCR or isothermal nucleic acid amplification. Alternatively, when the isolated nucleic acid targets is DNA no Reverse transcription is required and a specific PCR or isothermal nucleic acid amplification is performed using genomic or plasmid DNA. The (RT-)PCR conditions and reagents (in particular the primers and optionally probes) are selected to allow specific (RT-)PCR-based detection of target organisms of interest, e.g. to identify one or more sepsis- inducing microorganisms. The same applies also to isothermal nucleic amplification methods. When the genes conferring susceptibility or resistance to selected antibiotics are known, it is contemplated to use PCR as a means to detect those genes involved and thereby determine the targets' susceptibility/resistance to selected antibiotics and the like. The entire protocol from the sampling to the identification of bacteria/susceptibility testing usually takes about 14 to 8 hours. Above mentioned procedures for sample preparation and analysis can be performed (semi-)automatically with instruments using cartridges or bags loaded with required substances for processing target organisms proliferated in said liquid growth media. Another semi-automated method allows for direct use of 0.5-3.0ml of suspension with target organisms proliferated in said liquid growth media for sample preparation and analytics, e.g. DNA/RNA amplification and melting curve analysis. It is also possible to perform a parallel turbidity-based standardization of the cell count (e.g. McFarland) and subsequently an identification and/or antibiotic susceptibility according to standard protocols known in the art. It is further possible to perform biochemical analyses or additional antibiogram analyses in parallel, subsequent to the cell count determination.

Figure 26 (a+b) show a workflow embodiment of the present invention using (RT-)PCR for the identification of targets, e.g. bacteria suspected of causing sepsis and susceptibility testing thereof. First, the device of the invention is introduced for a period of about 5 min to 120 min, preferably for 30-60 minutes, into an environment in vivo or ex vivo suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria, particularly sepsis-inducing bacteria), e.g. a blood vein or a blood culture vessel. The targets are enriched on/in the device surface due to their specific adherence to binding partners that are attached to the surface of the inventive device. After removal of the device from the environment that is analyzed, the device is optionally subjected to at least one quick washing step to remove blood components that could interfere with detection. In one alternative, the bound target organisms are detached physically or chemically and transferred to a solid growth medium (e.g. a blood agar plate). In another alternative, the device with enriched targets is transferred into liquid growth medium (e.g. a Brain-Heart-Bouillon). The attached targets are detached using physical or chemical methods (e.g. using agitation, wiping off, or enzymatically or chemically detaching the bound targets from binding molecules, e.g. using enzymes, solutions having a shift in pH or ionic strength or the like) while the sampling device is in the vessel containing the growth medium and after detachment the sampling device is removed from the vessel. The liquid growth medium is incubated under suitable conditions for about 1 to 12 hours to allow target organisms to proliferate and multiply. After a sufficiently long incubation time of the growth medium to permit proliferation of the target organism, all or a part of the medium is removed to harvest the grown target organisms by means of centrifugation (according to standard procedures known to a person skilled in the art, e.g. a microbiologist). After removal of the supernatant, the pelleted target organisms are optionally washed and centrifuged again. A sample of the liquid culture is either directly transferred to a solid growth medium as in the first alternative) or it is first centrifuged and the pellet of target organisms washed according to standard protocol. In both alternatives, target organisms on the solid growth media are incubated for a sufficient time (e.g. overnight (e.g. 4-12 hours)) under suitable conditions to allow for growth of colonies. Subsequent to the cultivation step, colonies of the target organisms are picked (e.g. using a pipette tip) and transferred into a suitable buffer for lysis to release nucleic acids. Nucleic acids are isolated and purified using standard methods known in the art, e.g. in a diagnostic, molecular biology or clinical microbiology laboratory. The nucleic acids are subjected to Reverse transcription, if the target nucleic acid is RNA and then subjected to a specific PCR or isothermal nucleic acid amplification. Alternatively, when the isolated nucleic acid targets are DNA, no Reverse transcription is required and a specific PCR or isothermal nucleic acid amplification is performed using genomic or plasmid DNA. The (RT-)PCR conditions and reagents (in particular the primers and optionally probes) are selected to allow specific (RT-)PCR-based detection of target organisms of interest, e.g. to identify one or more sepsis-inducing microorganisms. When the genes conferring susceptibility or resistance to selected antibiotics are known, it is contemplated to use PCR as a means to detect those genes involved and thereby determine the targets' susceptibility/resistance to selected antibiotics and the like. The entire protocol from the sampling to the identification of bacteria/susceptibility testing usually takes about 13 to 16 hours. Above mentioned procedures for sample preparation and analysis can be performed (semi-)automatically with instruments using cartridges or bags loaded with required substances for processing colonies grown on solid growth media. Another semi- automated method allows for direct use of 0.5-3.0ml of liquid suspension of colonies grown on solid growth media for sample preparation and analytics, e.g. DNA/RNA amplification and melting curve analysis. It is also possible to perform a parallel turbidity-based standardization of the cell count (e.g. McFarland) and subsequently an identification and/or antibiotic susceptibility according to standard protocols known in the art. It is further possible to perform biochemical analyses or additional antibiogram analyses in parallel, subsequent to the cell count determination.

Figure 27 (a+b) show a workflow embodiment of the present invention using (RT-)PCR for the identification of targets, e.g. bacteria suspected of causing sepsis and susceptibility testing thereof. First, the device of the invention is introduced for a period of about 5 min to 120 min, preferably for 30-60 minutes, into an environment in vivo or ex vivo suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria, particularly sepsis-inducing bacteria), e.g. a blood vein or a blood culture vessel. The targets are enriched on/in the device surface due to their specific adherence to binding partners that are attached to the surface of the inventive device. After removal of the device from the environment that is analyzed, the device is optionally subjected to at least one quick washing step to remove blood components that could interfere with detection. The target organisms are either detached by physical methods or chemical methods and then subjected to nucleic acid isolation and purification steps, or the target organisms are first detached using physical or chemical methods and then subjected to centrifugation and washing steps, where after the pellet of centrifuged and washed target organisms is suspended and transferred into a suitable lysis buffer to release nucleic acids. Nucleic acids are isolated and purified using standard methods known in the art, e.g. in a diagnostic, molecular biology or clinical microbiology laboratory. The nucleic acids are subjected to Reverse transcription, if the target nucleic acid is RNA and then subjected to a specific PCR or isothermal nucleic acid amplification. Alternatively, when the isolated nucleic acid targets are DNA, no Reverse transcription is required and a specific PCR or isothermal amplification of nucleic acids is performed using genomic or plasmid DNA. The (RT-)PCR conditions and reagents (in particular the primers and optionally probes) are selected to allow specific (RT-)PCR-based detection of target organisms of interest, e.g. to identify one or more sepsis-inducing microorganisms. The same applies also isothermal nucleic acid amplification methods. When the genes conferring susceptibility or resistance to selected antibiotics are known, it is contemplated to use PCR as a means to detect those genes involved and thereby determine the targets' susceptibility/resistance to selected antibiotics and the like. In this embodiment it is possible to perform quantitative measurements of numbers of target organisms bound on a defined area on device using e.g. Q-PCR since said targets are not subjected to unspecific endpoint DNA/RNA amplification or to incubation on solid or in liquid growth media. The entire protocol from the sampling to the identification of bacteria/susceptibility testing usually takes about 3 to 8 hours. Above mentioned procedures for sample preparation and analysis can be performed (semi-)automatically with instruments using cartridges or bags loaded with required substances for processing target organisms which were directly detached from the device and transferred to suspension, e.g. PBS. Another semi- automated method allows for direct use of 0.5-3.0ml of suspension with target organisms which were directly detached from the device and transferred to suspension, e.g. PBS for sample preparation and analytics, e.g. DNA/RNA amplification and melting curve analysis. It is also possible to perform a parallel turbidity-based standardization of the cell count (e.g. McFarland) and subsequently an identification and/or antibiotic susceptibility according to standard protocols known in the art. It is further possible to perform biochemical analyses or additional antibiogram analyses in parallel, subsequent to the cell count determination.

Figure 28 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 10⁶ to 10² CFU/ml of E. coli strain K12 transformed with Green Fluorescent Protein (GFP).

Figure 29 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 10⁶ to 10² CFU/ml of E. coli strain K12 transformed with Green Fluorescent Protein (GFP).

Figure 30 shows the result of a qPCR-based detection of E. coli enriched in vivo for 15 min from a rat inoculated with E. coli transformed with Green Fluorescent Protein (GFP). Figure 31 shows an agar plate with colonies of E. coli transformed with Green Fluorescent Protein (GFP) 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 K12 transformed with GFP 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 32 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 circulation 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.

For all of the above inventive embodiments shown in the attached figures, it is contemplated to perform appropriate controls, i.e. to subject sample to respective analyses that have been treated essentially identical as the above workflows, except that the device of the invention is not introduced into an environment in vivo or ex vivo suspected of containing targets (e.g. target organisms such as microorganisms, for example bacteria, particularly sepsis-inducing bacteria), e.g. a blood vein or a blood culture vessel, but into an environment that should not contain any targets, e.g. sterile water, PBS or the like. Further, it is possible according to the invention to perform the above workflows in parallel, e.g. to grow colonies by inoculation of solid growth media with targets detached from an inventive sampling device, and to perform MALDI analysis using some of the colonies and in parallel, subsequently to, for example, a PCR-based analysis.

Detailed description of the invention

Before describing the invention in detail, it is deemed expedient to provide definitions for certain technical terms used throughout the description. Although the present invention will be described with respect to particular embodiments, this description is not to be construed in a limiting sense. Before describing in detail exemplary embodiments of the present invention, definitions important for understanding the present invention are given. Definitions

As used in this specification and in the appended claims, the singular forms of "a" and "an" also include the respective plurals unless the context clearly dictates otherwise.

In the context of the present invention, 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 %.

It is to be understood that the term "comprising" is not limiting. For the purposes of the present invention the term "consisting of is considered to be a preferred embodiment of the term "comprising of. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group which preferably consists of these embodiments only.

Furthermore, the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" etc. and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

In case the terms "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.

It is to be understood that this invention is not limited to the particular methodology, protocols, proteins, bacteria, reagents etc. described herein as these may vary.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention that will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. A "polynucleotide" or "nucleic acid" is a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single- stranded DNA and R A. It also includes known types of modifications including labels known in the art, methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as uncharged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), as well as unmodified forms of the polynucleotide.

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 (μ), delta (δ), gamma (γ), alpha (a), and epsilon (ε), 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 (K) and lambda ([lambda]) light chains. Within light and heavy chains, the 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. See generally, 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. For a detailed description of the structure and generation of antibodies, see Roth, D. B., and Craig, N. L., Cell, 94:41 1-414 (1998), herein incorporated by reference in its entirety. Briefly, the process for generating DNA encoding the heavy and light chain immunoglobulin sequences occurs primarily in developing B-cells. 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. In addition to the rearrangement of V, D and J segments, further diversity is generated in the primary repertoire of immunoglobulin heavy and light chains by way of variable recombination at the locations where the V and J segments in the light chain are joined and where the D and J segments of the heavy chain are joined. Such variation in the light chain typically occurs within the last codon of the V gene segment and the first codon of the J segment. Similar imprecision in joining occurs on the heavy chain chromosome between the D and JH segments and may extend over as many as 10 nucleotides. Furthermore, several nucleotides may be inserted between the D and JH and between the VH and D gene segments which are not encoded by genomic DNA. The addition of these nucleotides is known as N-region diversity. The net effect of such rearrangements in the variable region gene segments and the variable recombination which may occur during such joining is the production of a primary antibody repertoire.

The term "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.

The term "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 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.

"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. Immunol, 152: 5368 (1994)), single chain antibody fragments, diabodies (VH and VL domains on a single polypeptide chain that pair with complementary VL and VH domains of another chain) (EP 404,097; WO 93/1 1 161; and Holliger et al, Proc. Natl. Acad. Sci., USA, 90:6444-6448 (1993)), triabodies, tetrabodies, minibodies (scFv fused to CH3 via a peptide linker (hingeless) or via an IgG hinge), linear antibodies (tandem Fd segments (VH -CH1-VH -CHI) (Zapata et al, Protein Eng., 8(10): 1057-1062 (1995)); chelating recombinant antibodies (crAb, which can bind to two adjacent epitopes on the same antigen), bibodies (bispecific Fab-scFv) or tribodies (trispecific Fab-(scFv)(2)) (Schoonjans et al, J Immunol. 165:7050-57, 2000; Willems et al, J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci., 786: 161-76, 2003), nanobodies (approximately 15kDa variable domain of the heavy chain) (Cortez-Retamozo et al., Cancer Research 64:2853-57, 2004), an antigen-binding-domain immunoglobulin fusion protein, a camelized antibody (in which VH recombines with a constant region that contains hinge, CHI, CH2 and CH3 domains) (Desmyter et al., J. Biol. Chem., 276:26285-90, 2001 ; Ewert et al, Biochemistry, 41 :3628-36, 2002; U.S. Patent Application Publication Nos. 2005/0136049 and 2005/0037421), a VHH containing antibody, heavy chain antibodies (HCAbs, homodimers of two heavy chains having the structure H2L2), or variants or derivatives thereof, and 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. The term "monoclonal antibody" as used herein 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. ScLl U.S.A., 81 :6851 6855 (1984); Morrison and Oi, Adv. Immunol, 44:65 92 (1988); Verhoeyer et al, Science 239: 1534 1536 (1988); Padlan, Molec. Immun., 28:489 498 (1991); Padlan, Molec. Immunol, 31(3): 169 217 (1994); and Kettleborough, CA. et al, Protein Engineering., 4(7):773 83 (1991); Co, M. S., et al. (1994), J. Immunol 152, 2968- 2976); Srudnicka et al, Protein Engineering 7: 805-814 (1994); each of which is incorporated herein by reference in its entirety. One method for isolating human monoclonal antibodies is the use of phage display technology. 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. USA, 90:2551 (1993); Jakobovits et al, Nature, 362:255-258 (1993); Bruggermann et al, Year in Immuno., 7:33 (1993); WO 91/10741 , WO 96/34096, WO 98/24893, or U.S. Patent Application Publication Nos. 2003/0194404, 2003/0031667 or 2002/0199213; each incorporated herein by reference in its entirety.

Within the context of the present application relating to the capturing, enrichment, detection, identification and/or analysis of targets (cells and/or target molecules) involved in sepsis, the antibodies (or any derivative or modified form thereof) are specific for targets that are involved in sepsis, e.g. antibodies that bind specifically certain groups of bacteria (e.g. gram-negative or gram-positive bacteria), specific bacteria species or strains, fungi, yeasts, viruses, and the like.

"Epitope" or "antigenic determinant" refers to a site on an antigen present on a target cells or target molecule to which an antibody binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding (also referred to as discontinuous epitopes) are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8- 10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).

The phrase "specifically (or selectively) binds" to a capture molecule or "specifically (or selectively) reactive with", when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of an epitope, in a heterogeneous population of antigens and other biologies. Thus, 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. Similarly, 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.

The term "binding affinity" or "affinity" as used herein refers to the equilibrium dissociation constant (K_D) associated with each antigen-antibody interaction. In some embodiments, 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 ^{1 1} , 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 long period of time.

In the context of the present invention the term "target" relates to entire cells derived from the individual into which the sampling device or probe according to the present invention was introduced, or to microorganims, virus particles (virions), fragments thereof as well as molecules, such as polypeptides secreted from cells (e.g. interleukins, hormones, antibodies, LBP, (pro-)calcitonin, markers of inflammation, etc.), cell surface receptors, nucleic acids (DNA, RNA, microRNA, etc.), or sugar residues or polysaccharides (e.g. derived from glycoproteins, glyco lipids such as lipopolysaccharides or other gram negative bacterial-derived endotoxins, lipoteichoic acid derived from gram positive bacteria), derived from cells, microorganisms or viruses. In embodiments of the present invention, the targets are bacteria, fungi as well as molecules derived from bacteria or fungi. In embodiments of the present invention, the targets are bacteria, yeasts, fungi or molecules derived thereof, which may cause sepsis.

In the context of the present application, the targets bound to the probes used in context of the present invention may be detached using mechanical, physical, chemical, and enzymatic methods.

In the context of the present invention the term "sepsis" refers to an inflammatory condition caused by an infection with a microorganism, e.g. bacteria, yeasts, 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.

In the context of the present invention the term "target cell or microorganism inducing sepsis" relates to any microorganism that causes an infection that may induce blood- poisoning comprising, inter alia, staphyloccoci including Methicillin Resistant Staphyloccocus 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.

In the context of the present invention the term "probe" and "sampling probe" designates the part of the sampling device that is at least partially introduced into a sample container, a tube, or an individual whereof at least one clinical parameter should be determined, or wherein an infection should be diagnosed, etc.

In the context of the present invention the term "fibre" can designate both, optically transparent fibres for respective optical measurements of enriched target(s) 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. The fibres according to the invention may be coated with polymers, or they may consist of polymers. It is possible that the fibres according to the invention are hollow, e.g. polymeric fibres consisting of at least one polymer may be hollow.

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. Usually two fibre categories are discerned. "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. Furthermore, 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.

In other embodiments, 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 glass fibres are not coated with metals, particularly not with noble metals such as gold or platinum, which makes them less expensive and their production much easier. It is also possible to use fiberglass-enforced plastic fibres or Plexiglas (polymethylmethacrylate; PMMA) fibres and attach capture molecules directly or indirectly to their surface.

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.

The term "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.

The term "antibiotic compound" means an agent that either kills or inhibits the growth of a microorganism. Correspondingly, the terms "antifungal compound" and "antiviral compound" relate to agents that inhibit the growth or reproduction of fungi or viruses.

The term "capture elements" comprises molecules, for example antibodies, peptides, or aptamers, which are attached to the surface of the optical, e.g. silver halide fibre, or to polymer-coated glass fibre, and which are capable of binding specifically to other molecules, particles, or microorganisms of interest. The molecules may be modified chemically to optimize their biochemical parameters, and further 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. In some embodiments the antibodies bound to the polymer-coated fibres according to the present invention are specific for sepsis- inducing microorganisms, and preferably these antibodies are humanized antibodies.

The term "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 designates 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.

The term "microorganism" comprises single cell organisms such a bacteria, yeasts, fungi, 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 targets of interest, e.g. infectious microorganisms (bacteria, fungi, yeasts, viruses, etc.), or those that are known to have been exposed to certain targets, 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. patients who have a PCR- confirmed infection with HIV, but who show no clinical signs of (pre-)AIDS). The latter individuals with a confirmed diagnosis are also comprised by term "patient". The term "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.

The term "subject/individual a risk of development of a sepsis" refers to such organisms exposed or at risk of being exposed to sepsis-inducing organisms or molecules derived therefrom. For example, hospitalized patients, patients prior to or after surgery, 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.

The term "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. Often, 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 μιη while the core grains are larger than the cladding or surface grains.

The term „polymer" designates biologic and synthetic polymers that are capable of coating a glass fibre. 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. Photografting comprises techniques designated "photografting from" and „photografting-to". The longer and more spatially flexible the polymer chain, the higher is also the number of capture probes that can be immobilized on/in the polymer meshwork. Correspondingly, the likelihood of a larger number of bound targets increases with the number of capture probes. Examples of synthetic polymers are polyamine, polyamide, polyimine, polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyurethane, etc.

The terms "MALDI" and "MALDI-TOF" designate well established mass spectrometric techniques known to persons skilled in the art. In microbiology, MALDI/TOF spectra are used for the identification of microorganisms such as bacteria, yeast 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. In the context of the present invention, it is contemplated to provide a software database comprising MALDI-derived mass spectra that can be used as signatures, i.e. characteristic MALDI profiles, of sepsis-inducing cells or of molecules derived therefrom.

Below, embodiments of the invention are provided. It is noted that generally embodiments can be combined with any other embodiment of the same category (product, process, use, method).

In embodiments of the invention, a device for capturing, enrichment, detection, identification and/or analysis of a target, particularly a target cell or a component thereof capable of inducing sepsis is provided, characterized in that it comprises a sampling probe comprising capture elements specific for said target. In embodiments of the invention, the device for capturing, enrichment, detection, identification and/or analysis of a target, particularly a target cell or a component thereof capable of inducing sepsis, comprises a sampling probe comprising capture elements specific for said target, 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.

In embodiments of the invention, the device for capturing, enrichment, detection, identification and/or analysis of a target, particularly a target cell or a component thereof capable of inducing sepsis referred to above comprises a sampling probe comprising capture elements specific for said target, wherein the probe comprises a glass fibre, aluminium-coated glass fibre -, fiberglass-enforced plastic fibre, PMMA fibre, or the like.

In embodiments of the invention, the device according to any one of the preceding paragraphs comprises a glass fibre comprising a polymer coating or a metal- coating, preferably comprising at least one polymer selected from the group comprising synthetic or biological polymers, particularly polyethylene, polypropylene, polystyrene, polyamine, polyamide, polyacrylates, polyimine, polyester, polyacrylate,

polyetheretherketone, polyetherketone, polyurethane, polyvinylchloride, polymethylmethacrylate, polyethyleneterephthalate, Polytetrafluorethylene, chitin, to mention a few, or, for example, aluminium, copper, or gold as metal 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.

In other embodiments of the invention, the fibre consists of polymers, e.g. polymer selected from the group comprising synthetic or biological polymers, particularly polyethylene, polypropylene, polystyrene, polyamine, polyamide, polyimine, polyester, polyacrylate, polyetheretherketone, polyetherketone, polyurethane, polyvinylchloride, polymethylmethacrylate, polyethyleneterephthalate, Polytetrafluorethylene, chitin, etc.

In embodiments of the invention, the device according to any one of the preceding paragraphs with a target attached to or detached from 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), cell culture methods, and antibiograms. As known in the art, an antibiogram is tool for determining whether or not a given organism is susceptible or resistant to a given antibiotic active agent.

In an embodiment of the invention, 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.

In embodiments of the invention, the sampling device according to any one of the preceding paragraphs comprises a probe, particularly a fibre, wherein specific capture elements are disposed on the surface of the probe/fibre, particularly through a process called photografting, wherein said capture elements have at least one target specificity.

In embodiments of the invention, the sampling device according to any of the preceding paragraphs comprises at least two or more different specific capture elements a¹ , a²,...aⁿ 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 t¹ , t²,... tⁿ.

In embodiments of the invention, 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.

In embodiments of the invention, 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.

In embodiments of the invention as defined in the preceding embodiments, the capture elements are selected from the group comprising proteins, polypeptides, antibodies, nucleic acids, small molecules, aptamers, glyco lipids and lipopolysaccharides.

In embodiments of the invention as defined in the preceding embodiments, the probe is suitable for insertion into the body of an individual, wherein said individual is selected from the group comprising

individuals suspected to have been exposed to or known to have been exposed to a target of interest, wherein said target may further be selected from the group comprising bacteria, fungi, yeasts, viruses, parasites, and toxins, particularly those capable of inducing for a sepsis; individuals that are known to carry 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;

patients comprising human patients or veterinary patients;

patients undergoing in vitro-fertilization procedures.

In embodiments of the invention as defined in the preceding embodiments, the specific capture elements selectively bind bacterial cells or molecules derived from such cells.

In embodiments of the invention as defined in the preceding embodiments, the specific capture elements selectively bind bacterial cells capable of inducing sepsis in an individual or molecules derived from such bacterial cells.

In embodiments of the invention as defined in the preceding embodiments, the capture elements selectively bind bacteria selected from the group of Gram-positive and/or Gram-negative bacteria.

In embodiments of the invention as defined in the preceding embodiments, the capture elements selectively bind bacteria selected from the group comprising staphyloccoci including Methicillin Resistant Staphyloccucus Aureus (MRSA), streptococci, gram negative gastrointestinal bacteria, 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, and Bacillus cereus. Further, the capture elements may bind fungi such as those belonging to the genera Candida and/or Aspergillus.

Further embodiments of the present invention relate to methods 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 embodiments, further comprising quantifying the specific target, and optionally comparing the quantity with a threshold value. In embodiments of the above methods, specific targets are enriched.

In embodiments of the invention as defined in the preceding embodiments, the specific targets are bacterial cells or molecules derived from such bacterial cells, particularly those causing sepsis, optionally selected from the microorganisms referred to above. In embodiments of the invention as defined in the preceding embodiments, the sampling probe is introduced into an individual.

In embodiments of the invention, the enriched targets are provided in such form that may be transferred into or onto a culture medium for further cultivation. These targets may be detached from the probe using mechanical, physical, chemical or enzymatic methods. Alternatively, a part of the device used for enrichment in the methods of the invention may be cut of so that a part, or the entire probe carrying the capture molecules and targets is transferred as an element into or onto grow medium. Targets bound to the probe may thus grow from the probe into or onto a culture medium. It is also possible to detach the bound target using means that destroy the targets integrity so that they are no longer capable of growing. This can be done with harsh chemical or physical treatment. Respectively detached targets or parts thereof may be purified and/or used in, e.g. molecular biological methods, such as PCR. Such methods may require the purification and/or isolation of components of the targets such as nucleic acids or polypeptides prior to the step of subjecting the same to methods of characterization of the bound targets.

In embodiments of the invention, an in vitro method for the detection, analysis and/or quantification of specific targets in an enriched sample obtained from a patient is provided, wherein (i) the cell type and/or (ii) the molecular target structure and/or (iii) the quantity of obtained targets bound to a probe as defined in any one of preceding embodiments 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).

In embodiments of the invention, an in vitro method according to any the preceding embodiment is provided, further comprising (a) transferring selectively bound targets bacteria or fungi to a suitable growth medium in vitro, and (b) detecting, analyzing and/or quantifying the cell type or molecules originating from the obtained targets are determined. In option (a) of the above, the bound target cells that grow on or in a suitable medium are cultivated to further amplify their numbers. Subsequently, the target cells can be analyzed using one or more of several methods selected from staining methods (e.g. Gram staining), microscopic methods, e.g. using fluorescence microscopy after exposure of the target cells to staining procedures (e.g. fluorescently labeled antibodies and the like), antibiograms, detection of metabolites, molecular biologic methods (e.g. PCR), physico- chemical analysis procedures, spectroscopic methods, e.g. MALDI-TOF, etc.

In embodiments of the invention, an in vitro method according to any the preceding embodiments, the targets in an enriched sample are characterized using at least one method selected from the group comprising:

(a) microbiological methods, optionally an antibiogram for the identification of target cells,

(b) molecular biologic methods comprising methods for the characterization of the identity and/or quantity and/or mutational status of nucleic acids (e.g. PCR, RT- PCR and NGS) and/or polypeptides and/or glycoproteins or glyco lipids,

(c) microscopic methods comprising fluorescence microscopy, light microscopy, FACS, and/or

(d) physico-chemical or optical methods comprising Surface Plasmon Resonance methods, RAMAN spectroscopy, Infrared Spectroscopy,

(e) Mass spectroscopic methods comprising MALDI-ToF or LC-MS,

(f) 2d protein gel electrophoresis.

In embodiments of the invention, an in vitro method according to any the preceding embodiments is provided, wherein said method is followed by a methods 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. Currently available processes do not allow for a quantification of the targets, e.g. in the diagnosis of sepsis, do not allow determining the quantity of targets. Frequently, an obtained sample has to be subjected to a cultivation method, which leads to the proliferation of the targets in a more or less suitable medium under more or less defined cultivation conditions. The cultivation step and a subsequent analysis of the grown targets (e.g. bacteria) do not allow drawing conclusions on the amount that is actually present in the source of the sample. In other methods involving PCR, the obtained nucleic acids are first non- specifically amplified before a specific amplification step is performed. This makes statements on the amount of targets, e.g. bacteria, quite unreliable.

According to the present invention, it is possible to quantify the targets more exactly and respective methods are subject to the invention. Only a number of targets essentially corresponding to a given number of targets (taking into account the target size) can bind to the probe. If the period in which the probe is inserted in the medium that is analyzed for a defined time, and provided the area of the probe to which targets can bind is known, it is possible to quantify the number of targets that bind to a defined surface area over a defined period of time (expressed, e.g., as cfu/(mm² x min)). In some embodiments of the invention, wherein a monitoring of the number of targets over time is conducted (e.g. during therapeutic treatment) it is possible to control the effectiveness of respective therapy (for example when antibiotic treatment is performed and monitoring is conducted, e.g. over several days, the quantities may be determined after at least 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 24, 30, 36, 48 hours, etc.). Workflows of the invention that are exemplified by each of the figures relating to the invention may each comprise quantification steps.

In embodiments of the invention, a method of monitoring the presence and/or quantity of a specific target over time and/or at different loci is provided, 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 (t¹ to tⁿ), and/or comprising determining the quantity of a target at a first site (loc°) and at least one or more sites (loc¹ to locⁿ), wherein said target is selected from the group of pathogens (particularly those inducing sepsis), microorganisms, cells derived from the individual subjected to said method, molecules derived from pathogens (particularly those involved in the development of sepsis), 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.

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:

a) Providing a fibre,

b) Disposing capture molecules on the fibre surface or a layer of polymers deposited on the fibre, e.g. using photografting,

c) Optionally connecting the fibre with parts of the sampling probe, e.g. a holder

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 solutions, 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.

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.

Surprisingly, 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 such cells or molecules 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 target cells and/or target molecules 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 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 targets of interest in a subsequent application. For example, it is contemplated to use the device comprising a sampling probe according to the invention in the detection of a first target 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 target or a different target in a second patient. In some embodiments of the present invention, 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 capture molecules, proteins, for example mannose binding lectin and its engineered forms (Kang et al 2014), antibodies, peptides, or aptamers. In one embodiment, the sampling probe comprises a single silver halide fibre. In the context of the work underlying the present invention, it was surprisingly found out that silver halide fibres, e.g. 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. Further, even more surprising was the fact that capture elements as used herein may be immobilized directly on PIR fibres with additional noble metal, e.g., gold-layer as carrier substance. The latter fact substantially reduces the production costs for the herein described sampling probes. Further, 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. microorganisms inducing sepsis in a patient body, and/or molecules derived thereof. It was unexpectedly possible that the 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.

In an embodiment of the invention, the sampling device comprises a probe comprising at least one (optical) fibre, but it may also comprise at least two (optical fibres) fibres.

In embodiments of the invention, individual fibres are coated with, i.e. metals, colloidal metals, metal nanoparticles, grapheme, and/or some coated areas may be decorated with capture elements.

In embodiments of the invention, the device comprises a light source.

In embodiments of the invention, 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:

a) Providing a fibre, preferably a PIR fibre;

b) Immobilizing capture elements on said fibre;

c) Optionally assembling said fibre with additional components that enable the positioning, introduction, removal of the fibre from a patient and/or the combining of said fibre with components enabling the provision and measurement of optical signals;

d) Optionally providing a computer device and suitable connector devices, plugs or sockets optionally comprising software allowing for controlling, measuring, calculation, analysis, and interpretation of data obtained via the sampling probe.

In another embodiment of the present invention, the device comprises a sampling probe that comprises a polymer fibre, a fibre made of fiberglass-enforced plastic, or a Plexiglas-fibre. It was surprisingly found that 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. 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. It was unexpectedly possible that the 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 devices according to the present invention are further characterized in that they comprise a probe that is suitable for collecting, enriching, and/or analyzing targets (i.e. target cells and/or target molecules) through capture elements present at its surface that selectively and specifically bind the target.

In another embodiment of the invention, the sampling device comprises a probe comprising at least one plastic fibre, but it may also comprise at least two plastic fibres.

In another embodiment of the invention, 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.

In another embodiment of the invention, the silver halide fibre suitable for optical measurements comprises AgCl and/or AgBr. Preferably, the silver halide fibre is a PIR fibre. In an embodiment of the invention, the sampling device according to the present invention comprises a PIR fibre.

In an embodiment of the invention, the sampling device according to the present invention comprises a probe, which is an optical fibre.

In further embodiments of the invention, 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". Other 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. Of course it is also possible to use more than one fibre of each or both fibre classes (silver halide and plastic). It is not necessary that the same number of fibres of both classes is used in a mixed fibre, e.g. mixed fibres comprising 1 , 2, 3, 4, or more silver halide fibres may be combined with at least 1, 2, 3, 4 or more plastic fibres. It is contemplated that every possible combination of numbers of respective classes is encompassed by the term "mixed fibre". Furthermore, it is possible that the fibres have different diameters. In further embodiments, 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.). For example, when 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.

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. In embodiments of the invention, the individual fibres arranged in a fibre bundle are decorated with different capture elements, so that the entirety of targets bound or measured by the fibre bundle is greater or equal to the number or targets 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.

In embodiments of the invention, 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.

In embodiments of the invention, 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.

In embodiments of the invention, 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. In embodiments of the invention, at least one sharp element protruding from the tip or the surface of a fibre is rounded.

In embodiments of the invention, 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.

In an embodiment of the invention, the diameter of the fibre is < 1 mm. 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.

In an embodiment of the invention, specific capture elements having a least one specific binding activity for a target 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. In one embodiment, 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 targets. In other embodiments, 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.

In embodiments of the invention, 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 2.5mm or 1 mm.

In embodiments of the invention, 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.

In an embodiment of the invention, the sampling device comprises at least two or more different specific capture elements, which may be referred to as a¹ , a²,...a¹¹ 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 t¹ , t²,... tⁿ. For example, when it is desired to detect, enrich and analyze different bacterial species or strains, the sampling probe comprises corresponding different capture elements. Thus, when a probe according to the present invention is introduced into a patient in order to determine whether or not said patient has a bacterial infection, the capture elements (e.g. antibodies) may specifically detect LPS and/or lipoteichoic acid to thereby detect an infection with gram- negative and gram-positive bacteria, respectively. Further, 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.

In an embodiment of the invention, 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. Similarly, other molecules than antibodies can be selected to monitor the status of the patient over time, e.g. in terms of whether or not and to what extent such patient contains or produces targets 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 targets for autoantibodies, for example myelin, et cetera.

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. It is also possible to remove bound targets and further grow the same in suitable media, e.g. when the targets are cells, such as bacteria. Further, adherent bacteria may also grow on the fibre when suitable growth conditions are provided. Therefore, embodiments of the invention relate also to methods of culturing target cells previously enriched with the inventive probe fibre, wherein the probe fibre of a part thereof is transferred into/onto a suitable growth medium for these targets. Such media for bacterial growth are known in the field. They usually contain suitable carbon, nitrogen, oxygen, hydrogen, phosphorus and sulphur sources and a source of micronutrients (vitamins, minerals, etc.). Depending on the target cells, the target cell cultivation occurs essentially aerobically or anaerobically and using suitable physico-chemical conditions (e.g. pH-values, temperatures, etc.).

When the targets are molecules, it is also possible to perform additional methods to amplify the amount of target molecules, e.g. when the target is nucleic acid it is contemplated to use (RT-)PCR or cloning steps to produce more material for subsequent genetic analyses. Alternatively, it is possible to subject the molecules after removal to immune assays (e.g. ELISA, RIA, etc.), fluorometric, colorimetric, radioactive, or physico- chemical tests that permit the analysis of targets. 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 fragments thereof) carrying fluorescent moieties may be added to the bound targets 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 targets, as well as SPR.

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, 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. Respective diagnostic methods may rely on the detection of targets, e.g. of bacterial cells or molecules derived from such 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. In other words, 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 embodiments of the present invention, in vitro methods are disclosed, wherein obtained specifically enriched targets 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 targets remain bound to the sampling probe while non- specifically bound material is washed away as much as possible. Further, it is also possible to perform methods using the herein described device in extracorporeal material that was obtained from an individual, including artificial blood circulation systems (e.g. in dialysis).

In embodiments of the present invention, methods of selecting a suitable treatment of a patient are contemplated, 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. In a particular embodiment, 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. For patients that are carriers of sepsis-inducing targets, it can be advisable to monitor whether or not the target is still present during or after treatment, in particular, it is possible to quantify the targets, e.g. bacteria, over time according to the above described methods.

In further embodiments, 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. resistant to certain antibiotics (for example through establishment of an antibiogram), for example for antibiotic- resistant bacteria (MRSA, Klebsiella, and other microorganisms referred to e.g. in the claims) or whether an individual or patient, depending on its genetic set-up, may be or may not be treated with certain drugs, e.g. anti-cancer drugs (for example it is known that 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. Further, 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.

In some embodiments, 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 targets (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. In some embodiments, the diameter of the fibre is equal to or smaller than 1 mm, and preferably smaller than 500, 200, 125 μιη. In another preferred embodiment, 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. Furthermore, the cladding may include hole-like regions removed, which holes are partially decorated with capture elements. Finally 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. Furthermore, 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.

Furthermore, in 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.

In one embodiment of the present invention, excitation light is coupled into the fibre at an almost critical angle so that it travels to the distal tip in a "cladding mode". By means of 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. For example, 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 (FBGs) may be created in the fibre using, e.g., ultraviolet light. 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.

In one embodiment of the present invention 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. Between light source and 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).

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 18μιη 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. In another embodiment of the present invention, 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. In particular, 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.

As pointed out above, 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. For example, 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 ). 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. In this connection it is noted that the methods of collecting, enriching, detecting and analyzing targets not only concerns targets 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. In this connection it was surprising to be able to immobilize and analyze more than one target-specific capture element as well as the specifically captured targets using a given fibre or fibre bundle, wherein specific capture elements are immobilized at defined locations.

In embodiments of the invention, 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). In another embodiment, 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. In any of these embodiments, 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. As the coating of the fibre and the decoration of the metallic areas induces wavelength shifts on its own, so that SPR can be used also for the manufacturing and quality control of the device. In a preferred embodiment, 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.

Many types of excitation light sources can be integrated into the device, including LEDs Laser Diodes and quantum cascade lasers, C0₂ lasers alone, and all other lasers emitting between the UV and mid IR range of the optical spectrum. Several 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.

In other embodiments of the invention the device includes elements to measure FTIR (Fourier Transform Infrared Spectroscopy) or other implementations of Raman Spectroscopy. For this method, 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.

In embodiments of the invention, 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.

A specific version of Raman Spectroscopy is Surface Enhanced Raman Spectroscopy (SERS), which can also be employed with the device according to the present invention. For this method, 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. In this connection, it was challenging to provide sampling probes that are covered at least partially with a gold layer, wherein the gold-layer thickness surprisingly permitted analysis of targets using SERS. Once cells or molecules bind to the capture elements, 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.

In other embodiments of the present invention, fluorescence quenching is used to detect cells or molecules specifically. To this end, 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.

In other embodiments of the invention, Bio luminescence or Chemiluminescence are 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.

It is understood that the detection methods can be combined in the device. By employing mirrors, beam splitters or fibre couplers, excitation light from different sources can be coupled into the fibre. By using 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. In a preferred version, 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. In a preferred version, spectral separation is combined with array detectors.

In other embodiments, 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.

In other embodiments, 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.

The device according to the invention, 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.

In further embodiments of the invention, capture elements are attached employing a layer-by- layer electrostatic self-assembly (ESA) technology (Arregui et al 2010). For this method, 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).

In other embodiments of the device according to the invention, 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.

In further embodiments of the device according to the invention, 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.

In further embodiments relating to the device according to the invention, 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. After functionalization, the free carboxyl group can be used for conjugating capture elements, in particular bio molecules such as antibodies, peptides or oligonucleic acids covalently. Similarly, thiol containing ligand molecules may be attached to the silver ions.

Further methods were developed for functionalizing nanoparticles (Froimowicz et al. 2013). Many of them are not applicable for PIR fibres since these methods require integration of functional groups during synthesis of the particles, which is technically too burdensome with optical fibres. Fibres, instead, require functionalization post fibre synthesis.

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. In subsequent steps, 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.

Other suitable methods include using 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.

Further suitable methods comprise the deposition of 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.

The 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. Such hemocompatible coating may comprise particles or molecular layers or filaments, for example polyelectrolytes, polyethylenglycol (PEG) or even proteinaceous material like serum albumin.

Techniques for attaching the coatings to the device comprise those disclosed for capture elements, or combinations thereof. Sol Gel and hydrogel techniques may be employed as well. 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. Such localization elements are useful for the physician, assistant, or robot to navigate the fibre through the blood vessels of the patient. Furthermore, 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.

In further embodiments, 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 blood constituents using the device according to the invention, which 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 relate to uses in vitro.

In further embodiments of these method, 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. In embodiments of the method, 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. Through said catheter or cannula, 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. Once located, 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.

In further embodiments of the herein described methods, the online signal is used to control therapy parameters automatically. Information on specific targets, e.g. target cells such as pathogen, e.g. bacteria, or target molecules, e.g. antibiotics or other molecular concentration can be used to control the rate of medication released into the patient body. For example, 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. In other embodiments, 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. In other embodiments, 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.

It is also possible to reduce the rate of antibiotic relative to the concentration of toxins of pathogenic origin determined from the calculated signal, which could be generated by measuring the binding of said toxins to capture elements for toxins. It is possible to employ said signal to limit the toxic pressure induced by high numbers of dying pathogens by reducing the antibiotic release rate.

As pointed out above, once retracted, 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).

Being non-destructive, 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

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 polypropylene 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.

Subsequent to the removal of the enrichment probe from the artificial circulation system, 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 μΐ of ddH₂0 in Eppendorf tubes to elute bound target nucleic acids.

Subsequently, the presence of target miRNA was analyzed using a Stemloop

PCR analysis. Stemloop primers were used to provide cDNA and specific amplification was conducted by qPCR. Using a standard concentration curve, the amount of RNA originally present in the eluted samples in ddH₂0 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. Table 1 :

Regions of enrichment probes spotted with specific miRNA 122 capturing molecules (i.e. complementary antisense 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). Example 3 - Binding studies of miRNA 122-sequences (in form of DNA) in an artificial blood circulation system and in "dipping-experiments"

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

Overall the result shows that 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.

Example 4 - Binding studies using miRNA 122 (in form of RNA) in "dipping-experiments"

Phosphate buffer in tubes comprising synthetic miRNA 122 molecules at concentrations of 1 μΜ 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 R A target molecules is possible using the probes described above. The results are shown in Table 3.

Table 3

Example 5 - Enrichment of E. coli in "dipping", artificial blood circulation system and animal experiments

To show enrichment of E. coli on gold and polymer surfaces, dipping experiments were carried out in phosphate buffered saline (PBS) with E. coli added in different concentrations. The E. coli-dilutions were 10²; 10³; 10⁴; 10⁵ and 10⁶ colony forming units per ml (CFU/ml). Gold-coated aluminium-coated glass fibres as enrichment probes were functionalized with anti-lipopolysaccharide antibodies (anti-LPS-Ab WN1 222-5 obtained from the Forschungszentrum Borstel, Germany). The bifunctional crosslinker Dithiobissuccimmidylpropionate (DSP) was used for the functionalization of gold surfaces on the enrichment probes. For the functionalization of polymeric surfaces (polypropylene) a 3D- structure with free carboxyl-groups was applied. Subsequently, anti-LPS-Ab was covalently bound using conventional EDC/NHS chemistry. Partial functionalization of surfaces with different types of capture molecules was performed using the sciFLEXARRAYER System from Scienion AG. To this end, an enrichment probe was printed with different capture molecules labelled with different fluorescence molecules and the presence of successful immobilization of respective sample was checked using fluorescence detection using a fluorescence microscope.

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 „Pure Yield Plasmid Miniprep System" was used for DNA extraction. The amount of E. coli was determined by qPCR with specific primers against GFP.

The results of enrichment on the gold surface (A) and polypropylene surface (B) are shown in Figure 28.

To show enrichment of E. coli from entire blood, "dipping" experiments with human blood taken from blood conserves that were spiked with E. coli were carried out using the enrichment probe with gold surface as described above. Results are shown in Figure 29 (A).

Further, an artificial blood circulation system was used for binding experiments in flowing liquids. The enrichment probes were injected for 30 minutes into the system via a permanent venous catheter. E. coli-suspensions in PBS circulated with 20 ml per minute in concentrations described above. Results were shown in Figure 29(B).

For in vivo experiments the enrichment probes were inserted into the tail vein of rats via permanent venous catheters. An E. coli-suspension in PBS was inoculated via a port in the vena jugularis. The enrichment probes were removed after 15 minutes and processed as decribed above. qPCR-Results from animal experiments are shown in Figure 30.

In further animal experiments conducted as described above, 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 31. 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

To show enrichment of S. aureus on enrichment probes with gold surface combined with different detection were performed. S. aureus were diluted at a final concentration of 10⁶ 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-coated 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.

For cultivation experiments, the enrichment probes were scratched over an agar plate or inserted into liquid culture medium (brain-heart-bouillon or a blood culture medium). Experiments with S. aureus in PBS and blood, in "dipping" experiments as well as in artificial blood flow resulted in the growth of colonies (Figure 31) following an incubation of 12 hours at 37°C. Results from experiments in an artificial blood circulation system are shown in Figure 31 with colonies grown from enrichment probe scratched over an agar surface (Fig. 31, (A)) or colonies growing around a piece of an enrichment probe disposed on an agar surface (Fig. 31 (B)).

The identification of S. aureus enriched with the probes was performed with MALDI-Tof, qPCR and phenotypic characterization using a Vitek^® system (bioMerieux). Results from probes enriched from blood using the artificial blood circulation system and introduced into blood culture medium is shown in Figure 32.

Detachment of living S. aureus was carried out using a conventional supersonic cleaning bath for 10 seconds. Detached S. aureus bacteria were transferred to agar culture medium for cultivation over 12 hours. Results from a probe enriched from entire blood using an artificial blood flow are shown in Figure 33.

Further, it was possible to identify S. aureus detached from the probe according to the protocol in Example 5 and using specific qPCR.

Embodiments of the invention

1. 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.

2. The sampling device according to embodiment 1, wherein the probe comprises an optical fibre.

3. The sampling device according to any one of embodiments 1 and 2, wherein the probe comprises a silver halide fibre.

4. The sampling device according to any of the preceding embodiments, wherein the silver halide fibre comprises AgCl and/or AgBr.

5. The sampling device according to any of the preceding embodiments, wherein 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.

6. The sampling device according to any of the preceding embodiments, wherein the diameter of the fibre is < 1 mm.

7. The sampling device according to any of the preceding embodiments, wherein specific capture elements having a least one target specificity are disposed on the surface of the fibre.

8. The sampling device according to any of the preceding embodiments, wherein at least two or more different specific capture elements a¹ , a²,...aⁿ 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 t¹ , t²,... tⁿ.

9. 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 the fibre at different regions. 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 sampling device according to any of the preceding embodiments, wherein the capture elements are selected from the group comprising proteins, polypeptides, antibodies, nucleic acids small molecules, aptamers, small molecules, glycolipids, lipopolysaccharides. The sampling device according to any of the preceding embodiments, wherein the device comprises a light source. The sampling device according to any of the preceding embodiments, wherein said probe is suitable for insertion into the body of an individual, wherein said individual is selected from the group comprising individuals suspected to have been exposed to or known to have been exposed to a target, wherein said target may further be selected from the group comprising bacteria, fungi, viruses, parasites, and toxins; individuals that are known to contain 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 anticancer drugs and antibiotics;

patients comprising human patients or veterinary patients comprising mammalian patients;

female patients undergoing in vitro-fertilization procedures. The sampling device according to any of the preceding embodiments, wherein the specific capture elements selectively bind bacterial cells or molecules derived from such cells. 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 Staphyloccucus Aureus (MRSA), streptococci, gram negative gastrointestinal bacteria, 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, and Bacillus cereus. A method of detecting and/or analysing 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 targets. The method according to any one of embodiments 18 and 19, wherein the specific targets are bacterial cells or molecules derived from such bacterial cells. The method according to any one of embodiments 18 to 20, wherein the targets are bacterial cells selected from bacteria causing sepsis, optionally selected from the bacteria referred to in claims 16 and 17. The method according to any one of embodiments 18 to 21, wherein the sampling probe is introduced into an individual. An in vitro method for the detection, analysis and/or quantification of specific targets, wherein (i) the cell type and/or (ii) the molecular target structure and/or (iii) the quantity of obtained targets 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). The method according to any one of any of the preceding embodiments further comprising (a) transferring selectively bound targets bacteria or fungi to a suitable growth medium in vitro, and (b) detecting, analysing and/or quantifying the cell type or molecules originating from the obtained targets are determined. The method according to any of the preceding embodiments, wherein targets are characterized in combination with at least one method selected from the group comprising:

(a) microbiological methods, optionally comprising preparing an antibiogram and/or the identification of target cells,

(b) 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,

(e) Mass spectroscopic methods comprising MALDI-ToF or LC-MS,

(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 method according to embodiment 26, wherein 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 (t¹ to tⁿ), and/or comprising determining the quantity of a target at a first site (loc°) and at least one or more sites (loc¹ to locⁿ). The method according to embodiment 28, wherein 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 solutions, 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 target, particularly a target cell or a component thereof capable of inducing sepsis, characterized in that it comprises a sampling probe comprising capture elements specific for said target. The device according to embodiment 31, 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 according to any one of embodiments 31 and 32, wherein the probe comprises a glass fibre. The device according to any one of the preceding embodiments 31 to 33, wherein the glass fibre comprises a polymer coating, preferably comprising at least one polymer selected from the group comprising synthetic or biological polymers, particularly polyethylene, polypropylene, polystyrene, polyamine, and polyimine. The device according to any one of the preceding embodiments 31 to 34, wherein 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, quantitative PCR, isothermal nucleic acid amplification, cell culture methods, and antibiograms. The sampling device according to any one of the preceding embodiments 31 to 35, wherein the diameter of the fibre is < 1 mm. The sampling device according to any one of the preceding embodiments 31 to 36, wherein specific capture elements having at least one target specificity are disposed on the surface of the probe. The sampling device according to any of the preceding embodiments 31 to 37, wherein at least two or more different specific capture elements al , a2,...an 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 tl , t2,... tn. The sampling device according to any of the preceding 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 the preceding embodiments 31 to 39, 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 sampling device according to any of the preceding embodiments 31 to 40, wherein the capture elements are selected from the group comprising proteins, polypeptides, antibodies, nucleic acids, small molecules, aptamers, glycolipids and lipopolysaccharides. The sampling device according to any of the preceding embodiments 31 to 41, wherein said probe is suitable for insertion into the body of an individual, wherein said individual is selected from the group comprising individuals suspected to have been exposed to or known to have been exposed to a target, wherein said target may further be selected from the group comprising bacteria, fungi, viruses, parasites, and toxins; individuals that are known to contain 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 anticancer drugs and antibiotics;

patients undergoing in vitro-fertilization procedures.

43. The sampling device according to any of the preceding embodiments 31 to 42, wherein the specific capture elements selectively bind bacterial cells or molecules derived from such cells.

44. The sampling device according to any of the preceding embodiments 31 to 43, wherein the specific capture elements selectively bind bacterial cells capable of inducing sepsis in an individual or molecules derived from such bacterial cells.

45. The sampling device according to embodiment 44, wherein the capture elements selectively bind bacteria selected from the group of Gram-positive and/or Gram- negative bacteria. 46. The sampling device according to any of embodiments 44 and 45, wherein the capture elements selectively bind bacteria selected from the group comprising staphyloccoci including Methicillin Resistant Staphyloccucus Aureus (MRSA), streptococci, gram negative gastrointestinal bacteria, 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, and Bacillus cereus.

47. 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 embodiments 31 to 46, further optionally comprising quantifying the specific target, and comparing the quantity with a threshold value. The method according to embodiment 47 comprising enriching specific targets, particularly targets are associated with the development of sepsis. The method according to any one of embodiments 47 and 48, wherein the specific targets are bacterial cells or molecules derived from such bacterial cells. The method according to any one of embodiments 48 to 49, wherein the targets are bacterial cells selected from bacteria causing sepsis, optionally selected from the bacteria referred to in claim 15 and 16. The method according to any one of embodiments 47 to 50, wherein the sampling probe is introduced into an individual or an environment suspected of containing targets. The method according to any one of embodiments 47 to 51, wherein a sample of enriched targets is provided. An in vitro method for the detection, analysis and/or quantification of specific targets, wherein (i) the cell type and/or (ii) the molecular target structure and/or (iii) the quantity of obtained targets bound to a probe as defined in any one of embodiments 31 to 46 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). The method according to any one of any of the preceding embodiments 31 to 53, further comprising (a) transferring selectively bound targets bacteria or fungi to a suitable growth medium in vitro, and (b) detecting, analyzing and/or quantifying the cell type or molecules originating from the obtained targets are determined. The method according to any of the preceding embodiments 31 to 54, wherein targets 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 target cells,

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 glycolipids and/or identifying nucleic acids encoding for the susceptibility or resistance to antibiotics, - microscopic methods comprising fluorescence microscopy, light microscopy, FACS, and/or

physico-chemical or optical methods comprising Surface Plasmon Resonance methods, RAMAN spectroscopy, Infrared Spectroscopy, Mass spectroscopic methods comprising MALDI-ToF or LC-MS, 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 47 to 55, 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, optionally wherein said patient suffers from sepsis, further optionally, wherein the patient suffers from sepsis associated an infection with microorganisms whereof the susceptibility to or resistance to antibiotic compounds was determined. The method according to embodiment 56, wherein 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. The method according to embodiment 56, wherein the patient has a sepsis and 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 specific target over time and/or at different loci, comprising using the sampling device according to any one of claims 1 to 16 in a method according to any one of embodiment 47 to 58, further comprising determining the quantity of a target at a first point in time (to) and at least one or more points in time (ti to t_n), and/or comprising determining the quantity of a target at a first site (loco) and at least one or more sites (loci to loc_n). The method according to embodiment 59, wherein 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 method according to any of the preceding claims, wherein the device or probe as defined in any one embodiment 31 to 46 is used in a method in vivo or ex vivo for the enrichment of targets, optionally microorganisms, further optionally microorganisms causing sepsis, wherein the obtained targets are further subjected to any of the method steps as defined in Figures 4, 6-12, 14-20, and 22-27.

62. A kit comprising the sampling device or probe as defined in any one embodiment 31 to 46, 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.

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Claims

1. A method of enriching of a target indicating the presence or absence of bacteria and/or fungi causing sepsis in an individual or a target the presence or absence of which is implicated in the development of sepsis, comprising using a sampling device, wherein the sampling device comprises a sampling probe comprising capture elements specific for said target, wherein said sampling probe for use in vitro and/or in vivo is selected from the group comprising glass fibres, fully polymeric fibres and polymer-coated fibres, wherein said fibres may also be coated with metals selected from the group comprising gold, silver, platinum, and titanium, said method comprising

- providing a sample of enriched targets, wherein selectively bound targets are optionally transferred to a growth medium and cultivated,

- characterizing the targets using at least one method selected from the group comprising

(a) microbiological methods, comprising preparing an antibiogram, cell culture methods, determining the cell number, and/or the identification of target cells,

(b) 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 glycolipids and/or identifying nucleic acids encoding for the susceptibility or resistance to antibiotics comprising (RT-)PCR, quantitative PCR, isothermal nucleic acid amplification, Next-Generation Sequencing,

(c) microscopic methods comprising fluorescence microscopy, fluorescence-based methods, FISH, ELISA, light microscopy, FACS, and/or

(d) physico-chemical or optical methods comprising Surface Plasmon Resonance methods, RAMAN spectroscopy, Infrared Spectroscopy, ATR,

(e) Mass spectroscopic methods comprising MALDI-ToF or LC-MS, GC-MS,

(f) 2d protein gel electrophoresis,

wherein said methods may be performed consecutively or in in parallel.

2. The method according to claim 1, wherein said individual is selected from the group comprising - individuals suspected of having been exposed to or known to have been exposed to a bacterial target or fungal target causing sepsis;

- individuals that are known to contain a bacterial target or fungal target causing sepsis; and

human patients or veterinary patients.

The method according to any one of claims 1 and 2, wherein the bacteria are selected from the group of Gram-positive and/or Gram-negative bacteria, wherein said bacteria are selected from the group comprising staphyloccoci including Methicillin Resistant Staphyloccucus Aureus (MRSA), streptococci, gram negative gastrointestinal bacteria, 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, and Bacillus cereus, and wherein the fungi are selected from the group comprising those of the genera Candida, particularly Candida albicans, and Aspergillus.

A method of selecting a suitable treatment protocol of a patient suffering from a bacterial or fungal sepsis, comprising performing any of the methods referred to in the preceding claims, and selecting a suitable treatment for such patient depending on the identity, characteristics and/or quantity of the bacteria or fungi, optionally using further measurement data, optionally determining the susceptibility to or resistance to antibiotic compounds of the bacteria causing sepsis.

The method according to claim 4, wherein the treatment is selected from the group comprising treatment with antibiotics, fungicides, 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.

6. A method of monitoring the quantity of a bacterial or fungal target associated with sepsis over time and/or at different loci, comprising performing a method according to any one of the preceding claims, further comprising determining the quantity of a target at a first point in time (to) and at least one or more points in time (ti to t_n), and/or comprising determining the quantity of a target at a first site (loco) and at least one or more sites

7. A method according to any of the preceding claims, wherein the obtained targets are further subjected to any of the method steps as defined in Figures 4, 6-12, 14-20, and 22- 27.

8. A method according to any of the preceding claims, wherein the period from the provision of enriched targets to characterization of said targets takes from 5 minutes to 72 hours, from 10 minutes to 72 hours, from 15 minutes to 72 hours, from 30 minutes to 72 hours, from 45 minutes to 72 hours, or from 60 minutes to 72 hours, or from 1 to 60 hours.

9. A kit comprising the sampling device or probe as defined in 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.

10. A method according to any one of the preceding claims, wherein the sampling probe comprises at least two separate regions, wherein each region carries different types of capture molecules, and optionally at least one further negative-control region that does not carry and type of capture molecule.