WO2016016872A1 - A substrate and a method for purification and analysis of fluids - Google Patents

Abstract

A substrate for and a method to purify fluids, especially of biological origin, is provided here. The substrate has one dimensionally self-organized or self-assembled inorganic nanofibers. The fibers have diameter below 100 nm and ultra-high aspect ratio (length: diameter > 100,000:1). The substrate has a high porosity (over 50%) due to week bonds between adjacent fibers. The substrate is capable of withstanding temperatures over 350°C. A method to purify fluids is also provided.

Description

A SUBSTRATE AND A METHOD FOR PURIFICATION AND ANALYSIS OF FLUIDS

PRIORITY

This application claims priority of US provisional application US62/032015 filed on 1 August 2014.

Field of invention

The invention is related to materials solution used in separation of fluid components with the purpose of their simultaneous or consequent analysis. The separation methods based on pure filtration, Knudsen diffusion, adsorption, dialysis or osmosis are explicitly excluded. The analytical methods might be spectrometry, spectroscopy, etc. in any combination. One of particular example of the analysis methods is bio-analysis (analysis and separation of biological samples such as blood plasma, saliva or urine) with ultra-high pressure liquid chromatography (UHPLC).

Background of the invention

There is an increasing need of new bio-analytical methodologies with enough sensitivity, robustness and resolution to cope with the analysis of a large number of analytes in complex matrices in short analysis time. For this purpose, all steps included in any bio-analytical method (sampling, extraction, clean-up, chromatographic analysis and detection) must be taken into account to achieve good and reliable results with cost-effective methodologies [1].

Fast or ultra-fast separation methods are good tools to satisfy the necessity of reducing the total analysis time in bio-analysis where an increasing number and variety of samples is expected, and in areas where results must be obtained fast. Additionally, the number of target and non-target compounds is also increasing in some of these areas, especially when addressing drug development, abuse and threat substances, and doping control issues.

For instance, Japanese patent JP2004315331 discloses a filler for high-performance chromatography column as composite particles having the form wherein porous silica particles with the particle size of 500-20000 nm having ten or less micro pores with the holes diameter of 5-20 nm are bonded in a gourd shape. The goal of that invention is to provide a filler having a smaller pressure loss than a totally porous particle filler showing the theoretical plate height of the same degree, namely, a filler having improved separation impedance. Despite on claimed effects, it is admitted there that in order to improve the theoretical plate height, the particle size is required to be reduced, and, as the particle size is reduced resistance against the flow of a moving phase is increased and the pressure loss is increased.

US patent 8,021,967 discloses special patterned nanostructure to improve liquid transport method, based on wicking effect by arrangement of a nanoscale fibers allowing fluid to flow without any external power source. However, wick fluid transport is only possible if the fluid is wetting the solid surface to such extent that capillary pressure-driven transport greatly overrides other limiting factors such as Hagen-Poiseuille flow, inertia forces and viscous drag.

There are also several patents describing the application of nanofibers as adsorbents, acting either by physical forces or electrostatic potential differences. For example US patent 6,838,005 discloses aluminum hydroxide fibers of ~2 nm in diameter and with surface areas ranging from 200 to 650 m²/g, which are being made highly electropositive. When dispersed in water they are able to attach to and retain electronegative particles. This patent discloses that such electrostatic filters can be used for purification and sterilization of water, biological, medical and pharmaceutical fluids, and as a collector/concentrator for detection and assay of microbes and viruses. Macromolecules, such as proteins, may be separated from each other based on their electronegative charges. However, these claimed methods may not efficiently work if the substance of interest does not have necessary electric charge to interact with these nanofibers.

In general, application of nanofibers, especially natural (cellulose etc.) or synthetic (polymers) in testing and analytical devices is known. For example, US patent 7,998,748 generally outlines test elements, in particular diagnostic test elements, for determining the presence or concentration of biological, medical or biologically or medically effective substances including nucleic acids, proteins, viruses, microorganisms and cells, characterized in that these test elements contain nanofibers.

However, as shown in the present disclosure, mere presence of nanofibers does not itself guarantee good results in fluid separation and analysis. Accordingly, there is a need for alternative substrates and methods for purification and analysis of fluids, and especially those of biological origin.

Summary of the invention

Accordingly, in the present invention, the materials solution addresses problems in fast fluid components separation, and analysis is suggested to be composed of extreme ultra-high aspect ratio nanofibers having average diameters below 100 nm, preferably below 50 nm, with the ratio of length to diameter at least 100000:1. Furthermore, these nanofibers are also produced as a regular structure of co-aligned fibers, where nanoporous channels are self-established due to favorable weak bonds formation between the adjacent nanofibers. This results in open porosity fraction over 50%, preferably over 80%.

It is an object of this invention to provide a substrate and a method for purification of fluids.

It is an object of this invention to provide a substrate and a method for purification of fluids of biological origin, including but not limited to blood plasma, saliva and urine.

It is an object of this invention to provide a substrate and a method for purification of fluids for purpose of subsequent or simultaneous analysis of fluid components.

It is an object of this invention to provide a substrate and a method for rapid purification of fluids.

It is an object of this invention to provide a substrate and a method for purification of fluids where the substrate material can be heat sterilized and used multiple times.

It is an object of this invention to provide a substrate for miniature separation columns.

Short Description of Drawings

Fig. 1 is a scanning electron microscopy (SEM) photo of the fibers.

Fig. 2 shows results of RP-HPLC analysis of the analytes passed through the nanofibers column.

Detailed description of the invention

The rationale for the structure of the substrate disclosed herein evolves from the basics of the UHPLC method, although similar methodology is applicable for any such method where analytes separation proceeds in space and time. Fast chromatographic separations can be achieved either by increasing the mobile phase flow rate, by decreasing the column length or by reducing the column particle diameter [1]. Based on the Giddings [4] and Knox [6] interpretations, the efficiency expressed as the height equivalent to a theoretical plate (HETP) can be described as [1]:

where λ - structure factor, τ – obstruction constant, R _f - analyte retention factor, d – diameter of the packing material (particles or fibers), u – media velocity. It is important that media velocity (fluid rate) depends not only on imposed pressure drop, but also packing porosity (ε), medium permittivity (k), liquid viscosity and d values (at constant temperature). In turn, permittivity depends on porosity and d, for example via famous Carman-Kozeny equation [2] and the velocity for Hagen-Poiseuille flow [4,6,3]:

where N is the number of theoretical plates for the column of the length L. Any chromatographic column has its critical pressure, which is an intrinsic value meaning no chromatography separation can be achieved in finite time if the input pressure is lower than critical [4]. As permittivity k is also a function of d and the porosity, will be increasing with lower particle size. Thus most of the columns have particles diameter of 1.5-2.5 µm as otherwise the pressure drop would be too high to achieve reasonable fluid rates [1].

On the contrary to the packed columns with such fillers of any shape, the nanostructures of the present invention which as "naturally formed" with ultra-high aspect ratio and high porosity level, are capable of a substantial improvement in the UHPLC performance. For example, for the number of theoretical plates N = 10000 and for ~700 bar pressure drop the minimal achievable HETP size would be about 37 µm for 2 µm particles in the state of the art technology according to equations (1) and (2). For the nanostructure of the present invention with ultra-high aspect nanofibers of ~40 nm diameter at the same pressure drop, one may expect HETP ~10-100 nm i.e. approaching molecular-level resolution limit. In practical terms, this means a drastic decrease in separation time from 3-4 hours in the known systems down to seconds, allowing also substantial miniaturization of the UHPLC column down to millimeter-scales.

Among other parameters, which are important for such separation and analysis methods, besides minimal HETP and low separation times, is the reactivity of the substrate and the possibility of re-use of the column in the case of consequent tests (most of the multi-use columns have limiting temperatures of 60-90°C). This is of a particular importance for biological specimen, where certain disease-caused proteins (prions) and endotoxins are easily withstanding even dry heat sterilization at 200-250°C for hours. These temperatures are not suitable for organic materials (natural fibers, most polymers) and therefore such materials can be used only once. The most stable and inert materials towards such sterilization process (not involving chemicals or radiation) are inorganic materials, for example oxides, which can be utilized to produce nanofibers with ultra-high aspect ratio. Thus, it is reasonable to require that nanofiber materials in the substrate of the present invention must withstand dry heat sterilization cycles at temperatures above 350°C, where almost all known potentially harmful substances and those of interest (peptides, DNA, hormones, drugs) and living forms (bacteria, virions) can be destroyed in a convenient time scale.

To ensure selectivity of the substrate, these nanofibers can be additionally functionalized with a proper compound, or substance, or any other chemical, physical or topological methods (alike restricted access materials, synthetic antibodies, etc.), with necessary adjustment of the substrate hydro- or lyophilicity, which might not be constant along the thickness of the substrate (or the length of the column). Furthermore, such gradient modification of the substrate material, making it essentially a functionally gradated material (FGM), may allow construction of 3D substrate [5] with additional features depending on the desired application and the analysis goals.

In summary, the present invention discloses the substrate structure and the method of its application, which is capable of separation and/or analysis of the fluids, preferably of biological origin. The substrate is thus composed of the self-aligned, ultra-high aspect ratio (length: diameter > 100000:1) nanofibers (<50 nm diameter) with open porosity over 50%. The fibers are capable of withstanding high-temperature treatment over 350°C being made of inorganic materials such as oxides, which might be additionally functionalized to adjust their hydrophilicity or selectivity. The oxides may be selected from simple oxides, mixed oxides and oxide compounds. Simple oxides may be aluminum oxide, silicon oxide, titanium oxide or rare earth metal oxides, but other oxides may also be used. Mixed oxides may be Al₂O₃·SiO₂, but other mixed oxide may as well be used. Oxide compounds may be spinels or more complex oxides.

The nanofibers may be hydrophobic or hydrophilic. Optionally they may be functionalized with a third compound or molecule. Such compound or molecule may be an inorganic molecule, for example another oxide or material different from the substrate fiber material. Such molecule may also be selected from elements stable at the analysis conditions such as noble metals like Au, Pt, Ag, or C, or Si. Such molecule may also be an organic molecule selected from hydrocarbons, organic acids, aromatic compounds, and/or heterocyclic compounds. The organic compounds may contain one or more functional groups independently selected from amide, nitrate, carboxyl, hydroxyl and/or including halogen or chalcogen. The substrate described above could be also used as a scaffold for cultivation of living cells, bacteria, or any similar simple or combined in vitro studies.

The method of separation and/or analysis using abovementioned substrate includes contact and transport of the fluid in question through the substrate, where the fluid is preferably of biological origin, and where the functionalized substrate might be arranged to allow a functionally gradated 3D structure, which local reaction with analyte could be measured by any suitable technique on the spot (statically) or by scanning (dynamically) across the named substrate.

According to one embodiment of the invention the method comprises application of an analytical probe across thickness of the substrate and registering the probe signal, which is correlated with presence or concentration of a specific compound. The probe may be any known analytical method probe and the application of the probe maybe static (spot) or dynamic (scanning along the substrate thickness) mode. Examples of such probe might be spectroscopy (ultraviolet, visible or infrared range; Raman, etc.) or fluorescence, which application does not require explicit output of the analyte to come out of the column in time but rather by exploring the geometrical snapshot.

This substrate structure and the method of application allow substantial decrease of the separation and analysis time, miniaturization of the separation and analytical systems and improvement of the performance. The method used could be combined with other known analytical techniques.

Example 1

The ultra-high aspect ratio nanofibers, based on alumina compound, of average diameter of 40 nm (Fig. 1) were cut to the appropriate size (two pieces) to fit the „column“, which was a glass test tube. The weight of material was about 1.34 g. The material was tested to withstand at least 800°C, evidencing the benefit of the invention to enable multi-use columns for separation and analysis of biological fluids.

The analytes were chosen to have different polarity for comparison (phenol, pyridine and dimethylaniline). Standard solution of analytes was prepared in methanol with concentration 40 mM, which was diluted 10 times in toluene (final 4 mM concentration).

Fractions (0.5-1 ml) were collected in glass vials according to eluent compositions. The gradient was used from 0...20 % iso-propanol in hexane by 2% wt. steps. Collected fractions were analyzed at first on a TLC plate by spotting every fraction on plate and exposing it under UV-lamp (254 nm). Fractions that gave a visible spot were analyzed further with RP-HPLC (Agilent Technologies).

It was noticed that gravity pressure was sufficient to pass the analytes through the fiber column within a minute, which support the hypotheses above for only little pressure to be required in comparison with traditionally packed column which was unable to pass analytes at all without extra pressure.

The analytical results are shown in Fig. 2. It is noteworthy that simple ultra-high aspect ratio nanofibers, yet without any modification or functionalization, were able to pass analytes without extra pressure and nevertheless demonstrate retention and phase separation of compounds of different polarity. With a proper surface modification such columns will be tailored to specific compounds range according to the goals of the present invention.

REFERENCES:

[1] O. Núñez, K. Nakanishi, N. Tanaka, J. Chromatogr. A 1191 (2008) 231.

[2] P. Mirbod, Y. Andreopoulos, S. Weinbaum. J. Fluid Mech. 619 (2009), 147–166.

[3] J. van Brakel. Powder Techn. 11 (1975), 205-236.

[4] J.C. Giddings, Anal. Chem. 37 (1965) 6063.

[5] O. van der Biest, M. Gasik, J. Vleugels (eds). Functionally Graded Materials, TransTech Publ., (2005), 798 pp.

[6] J.H. Knox, J. Chromatogr. Sci. 15 (1977) 352-358.

Claims (16)

1. A substrate for fluids purification and analysis of fluid components, said substrate comprising one-dimensionally self-organized or self-assembled into the substrate during their manufacturing, co-aligned and adjacent inorganic nanofibers with diameters D less than 100 nm, and with ultra-high aspect ratio Length: D >100000:1 with open porosity over 50% and with weak bonds between each other thereby forming nanoporous channels in between the fibers.
2. The substrate of claim 1, wherein the diameter is below 50 nm.
3. The substrate of claim 1 or 2, wherein the open porosity is over 80%.
4. The substrate of any one of claims 1 to 3, where the nanofibers are made of inert materials, capable of withstanding temperatures over 350°C.
5. The substrate of any one of claims 1 to 4, wherein the nanofibers are made of oxides.
6. The substrate of claim 5, wherein the oxide is selected from the group consisting of simple oxides, mixed oxides, and oxide compounds.
7. The substrate of claim 6, wherein simple oxides are selected from Al₂O₃, SiO₂, TiO₂, and rare earth metal oxides, mixed oxides being such as Al₂O₃·SiO_2, and oxide compounds are selected from spinels (MgAl₂O₄) or more complex oxides.
8. The substrate of any one of claims 1 to 7, where the nanofibers are either hydrophobic or hydrophilic, or additionally functionalized with a third compound or a molecule, aimed on interaction with one or more components of the fluid.
9. The substrate of claim 8, wherein the third molecule is selected from an inorganic molecule, atom, or an organic molecule with one or more functional groups.
10. The substrate of claim 9, wherein the inorganic molecule is another oxide or material different from the substrate fiber material, the atom is selected from elements stable at the analysis conditions, such as noble metals like Au, Pt, Ag, and/or C, Si, the organic molecule is selected from hydrocarbons, organic acids, aromatic compounds, and/or heterocyclic compounds, and the functional groups are independently selected from amide, nitrate, carboxyl, hydroxyl and/or including halogen or chalcogen.
11. A method of fluids purification and simultaneous or consequent analysis, comprising the steps of:

    a) bringing the fluid into a contact with the substrate of any one of the claims 1 to 10, and

    b) allowing the fluid to transport along the direction of the organized nanofibers for a specified time.
12. The method of claim 11, wherein no additional pressure is applied in step b).
13. The method of claim 11 or 12, wherein the fluids are of biological origin, and wherein the method is also applied for the fluid selective fractionation without application of third chemicals or a thermal stimulus.
14. The method of claim 13, wherein the fluids are selected from the group consisting of blood plasma, saliva, and urine.
15. The method of any one of claims 11 to 14, further comprising static or dynamic application of an analytical probe across thickness of the substrate and registering a probe signal correlated with presence or concentration of a specific compound in the fluid.
16. The method of claim 15, wherein the probe signal is registered by spectroscopy or by fluorescence.

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