EP2206139A2

EP2206139A2 - Method and device for detecting at least one target substance

Info

Publication number: EP2206139A2
Application number: EP08843560A
Authority: EP
Inventors: Matthias Englmann; Philippe Schmitt-Kopplin; Istvan GEBEFÜGI; Heinz Frisch; Wolfgang Dietz
Original assignee: Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Current assignee: Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Priority date: 2007-11-02
Filing date: 2008-10-31
Publication date: 2010-07-14
Also published as: WO2009056327A2; DE102007052500A1; WO2009056327A4; US20110049354A1; WO2009056327A3

Abstract

The invention relates to a method for detecting at least one target substance, comprising the steps of converting molecules of at least one of the target substances to a gaseous state and a spectrometric detection of the molecules. The aim of the invention is to significantly increase the resolution of the method with regard to its detection limit and selectivity. The invention is characterized in that the conversion includes mixing/dissolving, forming an aerosol and evaporating at least one of the target substances with a solvent, the molecules being integrated into a gas phase, and the spectrometric detection includes an ionization of the molecules in the gas phase to give ions.

Description

Method and device for the detection of at least one target substance

The invention relates to a method and a device for the detection of at least one target substance according to the first and the eleventh patent claim.

In the case of detection of the aforementioned type, all or part of substances which can be converted from a mixture into a gaseous state as molecules are ionized and supplied to a subsequent detection in a mass spectrometer. Mass spectrometers are well known for the analysis of chemical substances from gases or dusts in various types.

From US 6,797,944 a method of laser desorption is known in which substances are formed by the action of a pulsed infrared light from a surface for transmission to a chemical analysis system, e.g. a mass spectrometer can be desorbed molecularly or atomically. Due to the pulse length and pulse repetition rate, certain substances can be selectively desorbed.

Furthermore, WO05 / 047848 describes a process in which a solution with a target substance is evaporated in a microchannel structure and supplied with a carrier gas for ionization of a corona zone. This is followed by detection of the ions.

However, detection of a large number of target substances in a mixture requires not only a significantly increased selectivity of the method used, but also extended detection limits.

Based on this, the object of the invention is to propose a method and an apparatus for the detection of at least one target substance, which differs from the prior art by a significantly increased resolving power in the detection limit and in the selectivity distinguishes.

The object is achieved with a method and a device having the features of claims 1 and 11, respectively. The referenced to this subclaims give advantageous embodiments again.

The invention relates to a method for the detection of at least one target substance and to an apparatus for carrying out the method.

The method comprises a transfer of molecules of at least one of the target substances into a gaseous state and a subsequent spectrometric detection of the molecules, preferably by means of a mass spectrometer.

An essential feature is that the transfer of the molecules comprises a soluble mixing, an aerosol formation and evaporation of at least one of the target substances with a solvent, wherein the molecules are integrated into a gas phase.

The soluble mixing of the molecules comprises in solution bringing the target substance or substances into a solvent, which presupposes a solubility of the target substance with the solvent in the liquid and / or gaseous state. Only within the scope of an advantageous embodiment does this exclude emulsifying or dispersing a portion of the target substance into the solvent. In that case, only a selective soluble mixing of a part of the target substance takes place, while the remaining other part of the target substances is insoluble in the solvent and consequently does not become molecularly distributed in the solvent. In a further preferred form, a targeted utilization of temperature-dependent solubilities of a target substance with the solvent takes place, with an admixture of this target substance in the solvent being determined solely by the selection and setting of a specific mixing temperature. between a soluble and an insoluble, eg emulsifying interference.

A further advantageous embodiment comprises a soluble mixing of one or more target substances with the solvent in the presence of an additional carrier substance, wherein the carrier substance is preferably soluble as a particle or as a liquid in the solvent and adsorbs the molecules. The adsorption preferably takes place before the mixing. During the mixing, the adsorbed target substances are then transported via the dissolving carrier substances in the solvent, homogeneously, preferably distributed as molecules or molecular groups, and thus in the case of insolubility with the solvent, ideally molecularly mixed.

Mixing is preferably carried out continuously by combining the target substances and the solvent. Micromixers, disclosed by way of example in DE 199 28 123 A1, advantageously promote continuous spontaneous simultaneous mixing of two liquids.

Furthermore, an additional equipment of the mixing device with a separation device such. HPLC (high performance liquid chromatography apparatus) or electrophoretic separators (e.g., based on capillary electrophoresis) for separating a plurality of target substances before or after mixing with the solvents is an additional embodiment.

The invention also includes the use of a plurality of solvents, wherein preferably one or more target substances are mixed separately into each solvent and the resulting solutions are then combined.

Together with or after the soluble mixing takes place an aerosol formation in which the target substances are atomized with the solvent (s) to form an aerosol. Another essential feature of the invention comprises aerosol formation by an aerosol former. Preferably, this is done by droplet formation by means of a dispenser, wherein a predetermined number of droplets preferably with the same droplet size (10 to 200 pL, preferably 20 to 100 pL, more preferably between 30 and 80 pL, more preferably 40 to 60 pL volume) and substance mixture ratio ( Target substances and solvents) can be produced, or together with a mixing by means of a two- or multi-fluid nozzle. Dispensers are suitable both for atomization of the solution after mixing as well as by separate atomization of the solvent to be mixed and target substances in a common aerosol cloud. It is within the invention to promote the formation of aerosol by additional measures on the aerosol, for example by applying the liquid or solution to be dispersed with ultrasonic waves or electric charges (Elekt- rospray), wherein similarly electrically charged liquid particles not only repel each other, but also be electrically attracted in an electric field to a counter electrode such as by a heating element in the aforementioned evaporation device.

Alternatively, aerosol formation can also be achieved by means of bubble bursting, in which a bubbling, boiling or otherwise gas bubble-forming liquid comprising solvent and all or only some of the target substances are arranged in an open vessel. The forming gas bubbles rise to the liquid surface and burst there, which are released by the thereby relaxing bubble surface aerosol droplets. The target substances in the liquid mix in the formation in the gas volumes or at the adjacent to the gas volumes liquid interfaces of the bubbles and are released when bursting out there from there with the solvent as aerosol drops in the ambient atmosphere. Additional substances in the solution, such as surface-active substances (eg surfactants, foaming agents) with possible structure-specific affinities for the target substance influence or promote a selective concentration of the target substance in the bubbles and in the droplets that arise during bubble bursting. Likewise, an optional direct evaporation of the foam on the preheated heating element surface can also be used for targeted enrichment and measurement of the target substances.

Another essential feature of the invention comprises evaporation of the solvent present as an aerosol with the target substance (s). The evaporation is preferably carried out thermally on a Heizelementoberfläche with a surface temperature preferably above the boiling temperature of the solvent, wherein the target substances are preferably transported molecularly from the solvent gases and spread in gaseous form. If the surface temperature is below a boiling temperature of one of the target substances, aerosol fractions (i.e., not single molecules or molecular groups) from that target substance are selectively or not significantly significantly evaporated more slowly, e.g. on the Heizelementoberfläche and are kept away or separated in this way from the forming gas phase. This effect can be achieved e.g. also for a selective enrichment of a specific target substance on the Heizelementoberfläche exploit. By pulsed heating for evaporation, the enriched target substances are convertible into the gas phase and thus are advantageously in concentrated form for further analysis, e.g. available in a mass spectrometer. In this way not only the detection limits of certain target substances can be shifted downwards, but also a material separation of target substance groups, in particular with many target substances, can be realized.

Increased integral or selective adhesion to at least one of the target substances is achievable by treatment or coating of the heating element surface. For example, a functional coating with nanoparticles or a polymer adsorption coating (containing or consisting of nanoparticles) can be used. Kel or chemical polymer adsorption coating) to a concentration of the target substances with an increased adsorption tendency. The target substances concentrated over a certain time can also be detected quantitatively on the coated or treated heating element surface as a closed sample by a further analysis method.

Evaporation of an aerosol, which has arisen due to bursting of gas bubbles rising in liquid, is preferably carried out by means of a heating element arranged above the liquid surface. The heating element surface is preferably arranged horizontally.

Another embodiment comprises an open-pore heating element, wherein the aerosol passes through the open pores and is thereby evaporated. The open porosity form the heating capillaries whose walls represent the Heizelementoberflächen and possibly in o. G. Senses are coated or treated. By a vacuum suction, the aerosol is sucked through the heating capillaries. In the embodiment with an aerosol formation by ascending bursting gas bubbles, the open-pore heating element is preferably arranged plate-shaped above the surface of the liquid.

The invention further comprises ionization and means for ionizing molecules or molecular groups of the target substance in the gas phase into ions. The ionization is preferably carried out as photoionization, preferably with a laser light VUV or UV source. In a preferred embodiment, the laser light VUV or UV source serves not only for photoionization, but also for integral or local heating of the heater surface, either as a single or as an additional energy source.

Furthermore, the invention comprises a spectrometric detection as well as a mass spectrometer for carrying out this detection. It is within the scope of the invention to use the method and apparatus for the quantitative detection of certain biological or biochemical substances such as axeropthenes, retinols, terpineols, citrals, geranylacetates, nootkationse, bisabolenes or decanes as the target substance in absolute form or from a substance mixture, wherein the Heating element surfaces can also be formed by natural or processed sample surfaces up to parts of the carcass or tissue samples and can be heated by light irradiation, for example. Evidence includes in vitro studies of body fluids as well as in situ studies.

In the following the invention will be explained in more detail with reference to embodiments and figures. Show it

Fig.l a first embodiment with uncoated heating element,

2 shows a second embodiment with coated heating element,

3 shows a third embodiment with rising and bursting on a liquid surface gas bubbles for aerosol formation,

4a to c further embodiments with Ansaugkapillaren to a mass spectrometer,

5 shows an embodiment with an evaporation device with laser scanner and

Figure 6 is a determined within the scope of the invention mass spectrum for a drop of a 1 mg / L solution of D10-pyrene in methanol and

7a to c are each a determined within the scope of the invention mass spectrum of a 10 mg / L HSL standard solution at 80 ⁰ C (a), 100 ⁰ C (b) and 102 ⁰ C (c) evaporation temperature. As illustrated in FIGS. 1 to 5, the exemplary embodiments of a device for the detection of at least one target substance comprise a dispenser 1 (FIGS. 1, 2, 4 and 5) or a gas bubble 6 forming liquid 7 (FIG. 3) as aerosol former , which is aligned with its main radiation direction 2 of the aerosol on the Heizelementoberfläche 3. When the aerosol hits the heating element surface 3, a gas phase cloud 4 is formed by evaporation, which is then irradiated with a light beam 5 from a laser, UV or VUV source 8 and ionizes the molecules of the target substances. The ionized molecules are withdrawn from the gas phase cloud and forwarded to a mass spectrometer 11

Fig. 2 shows by way of example a coating 9 on the heating element 10, e.g. one of the abovementioned functional coating with nanoparticles (compare FIGS. 4 c and d). The heating of the heating element surface 3, i. the surface exposed to evaporation for the aerosol is thus indirectly through the coating.

Fig.l, 4a, 4c and 5 show embodiments in which the light beam 5 is directed to the Heizelementoberfläche 3 and is heranziehbar as a separate or additional heating for the evaporation. In these cases they are also part of the evaporation device.

5 shows an exemplary embodiment in which the light beam 5 follows a cell-shaped scanning movement 12 on the heating element surface and thus, with time resolution, brings only the substances which are directly irradiated with solvent onto the surface regions for evaporation. Such an embodiment is preferably suitable for Adsorptionsuntersuchungen of target substances on natural or post-processing surface with several different surface areas as Heizelementoberfläche. The heating of the heating elements is preferably carried out exclusively by the light beam. - S -

4 a to c show by way of example devices in which the molecules are aspirated in the gas phase through a capillary 14 and forwarded to the mass spectrometer 11. 4a (see also Fig. 5) shows an embodiment in which the capillary terminates in the gas phase cloud 4, preferably at or as close as possible to the point on the heating element at which the gas phase is formed by evaporation. FIG. 4 b shows by way of example an embodiment with evaporation chamber 13 (closed system) in contrast to all other systems shown. Further, on its way to the mass spectrometer 11, the capillary 14 may be separated from an ionization chamber 15 with ionizing means, e.g. a laser, UV or VUV source 8 (see Fig. 4b) and / or with a GC capillaries 16 (see Fig. 4c) be provided. A series connection of the two aforementioned ionization chamber and GC Kapiilare in the capillary is basically possible in any order. However, the effectiveness of a GC capillary is also dependent on ionization of the substances passed through, with reproducibility of a substance separation being ensured, in particular in the case of non-ionized substances. Therefore, a preferred embodiment comprises a capillary 14 with integrated ionization chamber 15 and GC capillary 16, wherein the ionization chamber downstream of one or more GC capillaries and the mass spectrometer 11 is connected directly upstream (see Figure 4d).

In principle, however, it is also an ionization of molecules in two places, i. such as. both on the heater surface (see Figures 1, 2, 3, 4a, 4c and 5) and in a separate ionization chamber (see Figures 4b and 4d), especially in combination with other separation devices, e.g. a GC capillary can be used to optimize the selectivity of the method for specific target substances.

6, 7a to c and 8 give by way of example mass spectra, ie the intensities 18 plotted against the ratio of mass to charge 17 again, as determined in experiments within the scope of the invention. The solvents used for the experiments are commercially available products of analytical quality. The spectrum shown in FIG. 6 reflects the sensitivity of the method. It was according to a device. Fig.l, with laser ionization ie with an evaporation of single drops (drop volume 52.5 pL) determined by a dispenser on an uncoated heating element surface. The result represents the resolution of a single drop of a solution of 1 mg / L DlO pyrene in methanol as peak 19. Even with the evaluation of a drop, this peak stands out significantly over the signals surrounding it; the method has a high sensitivity, ie a low detection limit.

A determination of a spectrum gem. FIG. 6 can be considerably accelerated with a rapid separation with a UPLC system (Ultra Performance Liquid Chromatography) as follows. Starting product was a mixture of several polyscyclic aromatic hydrocarbons (PAH), which was initially supplied to the UPLC separation. The separated PAH to be tested are then mixed in a ratio of between 30 and 100% in acetonitriles at a flow rate of 0.9 mL / min in 0.5 min, followed by an isocratic flow (100% acetonitrile) of 0.2 min for homogenization , In this way, a D10-pyrene fraction of 52.5 pL (1

Drop) from a total fraction of the starting product of 1080 μl, with e.g. dosing with a dispenser, ionizing with a UV source of the type mentioned in the beginning, detecting with an ICR-FT mass spectrometer and measuring a spectrum acc. Determine Fig.6.

The aforementioned procedure is characterized by a careful task and treatment of the biomolecules on the one hand and the isolation of the substance in a very short time on the other hand and thus also allows the detection, for example, also atmospheresensible or otherwise sensitive substances for the identification of metabolites (Metabolomics), respiratory condensates , Cerebrospinal fluid (cerebrospinal fluid) or microbiopsy specimens. The risk of thermal decomposition during ionization of the molecules is thus benso reduced as a fragmentation of the substances to be examined. The generally small sample volume required for a reliable analysis enables important future applications such as microtechnical analysis systems (LabOnChip), for example, a substance separation in fluidic chips in a confined space or with large throughputs for the isolation of trace constituents with very low concentrations. In particular, the very high separation efficiency in a very short time and the required need for analytes after the separation in the picoliter range are advantageous.

FIGS. 7a to 7c, on the other hand, show spectra, likewise determined in a device according to FIG. Fig.l, but with UV ionization. As evaporation temperatures on the heating element surface 80, 100 and 12O ⁰ C were selected (see Fig.7a, b and c). The solution used as the model substance consisted of 10 mg / L N-acyl homoserine lactone (HSL) with carbon chains with 4, 6, 8, 10, 12 and 14 carbon chain length (in Figure 7a to c as c4 to cl4) in methanol , where the proportions of the respective chain lengths in the solution were identical in all three experiments. HSL are signal substances that play an important role in the inter-bacterial communication of some bacteria. In the determined spectra, a selectivity of the HSL given by the temperature of the heating element surface is clearly recognizable as a function of the chain length. While short-chain HSL, in particular the C4 and C6-HSL outweigh predominantly at evaporation temperatures up to 100 ⁰ C (see FIG. 7a and b), they occur at 12O ⁰ C compared to the longer-chain cl2 and C14-HSL in the background (see FIG. Fig.7c). While C14-HSL practically at 80 ⁰ C does not occur (see FIG. 7a) in appearance, it forms the highest peak at 120 ⁰ C. This experimental example illustrates the possibility of controlling the selectivity using the example of a temperature dependence of a solution with several target substances. Higher temperatures increasingly evaporate the larger molecules of more polar target substances, while shorter-chain target substances have lower thermal stability and lower temperatures evaporate. This selectivity can also be used to confine initially unknown target substances in a solution.

List of reference numbers:

1 dispenser

2 main beam direction

3 heating element surface

4 gas phase cloud

5 light beam

6 gas bubbles

7 liquid

8 VUV source

9 coating

10 heating element

11 mass spectrometers

12 Scanning motion

13 evaporation chamber

14 capillaries

15 ionization chamber

16 GC capillaries

17 Ratio of mass to charge

18 intensity

19 peak of DlO Pyrene

Claims

Claims:

1. A method for the detection of at least one target substance, comprising a) a transfer of molecules of at least one of the target substances in a gaseous state and b) a spectrometric detection of the molecules, characterized in that c) the transfer of a soluble mixing, aerosol formation and evaporating at least one of the target substances with a solvent, wherein the molecules are integrated into a gas phase, and d) the spectrometric detection comprises ionization of the molecules in the gas phase into ions.

2. The method of claim 1, wherein the mixing with the solvent is selectively carried out only a part of the target substances, wherein the remaining part of the target substances is insoluble with the solvent.

3. The method of claim 1 or 2, wherein the molecules of the target substance are mixed before mixing with the solvent with an additional carrier substance, wherein the carrier substance is soluble with the solvent and adsorbs the molecules.

4. The method according to any one of the preceding claims, wherein the evaporation takes place on a Heizelementoberfläche with a surface temperature above the boiling temperature of the solvent.

5. The method of claim 4, wherein the surface temperature does not exceed the boiling temperature of at least one target substance.

6. The method of claim 4 or 5, wherein on the Heizelementoberfläche at least one of the target substances is bound by an increased adhesion tendency.

7. The method according to any one of the preceding claims, wherein all or only part of the target substances is dissolved in a liquid as a solvent in which gas bubbles form with gas volumes, the target substance in the formation in the gas volumes or adjacent to the gas volumes liquid interfaces mixes and the aerosol formation takes place by bursting of the gas bubbles on the liquid surface.

8. The method according to any one of the preceding claims, wherein the ionization by means of a photoionization during and / or after the evaporation takes place, wherein the target substances and the solvent are irradiated immediately before and after the evaporation of a laser light, VUV or UV source ,

9. The method of claim 8, wherein the required for the evaporation of energy input by the laser light, VUV or UV source.

10. The method according to any one of the preceding claims 1 to 7, comprising forwarding the evaporated target substances and the solvent in an ionization chamber in which the target substances and the solvent are ionized irradiated only after evaporation from a laser light, VUV or UV source ,

11. A device for detecting at least one target substance for carrying out a method according to one of the preceding claims, comprising a) a mixing device, an aerosol former (1) and an evaporation device (3, 10) for the solvent and molecules of at least one of the target substances, b) a laser light, VUV or UV source (8) for photoionization of the molecules into ions and c) a mass spectrometer (11) for the spectral detection of the ions.

12. Device according to claim 11, wherein the mixing device and the aerosol former comprise a two-substance nozzle or a dispenser (1).

13. The apparatus of claim 11, wherein the mixing device and the aerosol former comprises a device for aerosol formation by bubble bursting.

14. Device according to one of claims 11 to 13, wherein the evaporation device comprises a Heizelementoberfläche (3).

15. Device according to claim 14, wherein the heating element surface (3) has a functional coating (9) with nanoparticle or a polymer adsorption coating for concentration of target substances.

16. Device according to one of claims 11 to 13, wherein the evaporation device comprises a natural or a machined surface of a sample with heating means.

17. The apparatus of claim 16, wherein the heating means comprise a laser light, VUV or UV source (8).

18. Device according to one of claims 11 to 15, wherein the mass spectrometer has a directed to the evaporation device suction capillary (14).

19. The apparatus of claim 18, wherein the suction capillary (14) comprises a gas chromatograph (16).

20. The apparatus of claim 18 or 19, wherein the Ansaugkapillare

(14) opens into a closed ionization chamber (15) with the laser light, VUV or UV source (8).

21. Device according to one of claims 11 to 20, wherein the mixing device comprises an HPLC or an electrophoretic separation device.