WO2011007139A2

WO2011007139A2 - Method and apparatus for determining analyte parameters or recording analyte information.

Info

Publication number: WO2011007139A2
Application number: PCT/GB2010/001344
Authority: WO
Inventors: David R. Klug; Christian Bernd Loeffeld
Original assignee: Imperial Innovations Ltd
Current assignee: Ip2ipo Innovations Ltd
Priority date: 2009-07-14
Filing date: 2010-07-14
Publication date: 2011-01-20
Anticipated expiration: 2012-01-14
Also published as: GB0912230D0; WO2011007139A3

Abstract

A method is provided for determining an analyte parameter. The method comprises measuring a plurality of values for electrophoretic mobility of the analyte for a plurality of electric field strength. The method further comprises determining a coefficient of a relationship between mobility and applied electric field strength and deriving a parameter from that coefficient.

Description

Method and apparatus for determining analvte parameters

or recording analyte information

The method relates to an invention or apparatus for determining analyte parameters or recording analyte information.

Analyte mobility separation processes such as electrophoresis are well known. One such process is capillary zone electrophoresis, an apparatus for which is shown in Fig. 1. The system designated generally 100 includes a source of sample or analyte in a buffer solution 102 at a first electrical potential in communication with a capillary 104. An electric potential is applied across the capillary.

The mobility of the analyte is most generally defined by the Henry-modified Debye-Hueckel relation,

(Ia) μ_o = (2εζ/3η)- f(κ - a)

Where e is the dielectric constant of the buffered system and ζ the zeta potential of the analyte. Equation (Ia) converts into the Helmholtz-Smoluchowski relation μ₀ - (εζ/ η) for the Henry function ϊ(k.a) converging to 3/2 for high electrolyte concentrations. Alternatively, the mobility can be described via the Henry-modified Einstein relation

(_lb) μ_o = (q/6πηR)' f(κ - a)

Where z is the charge, r is the diameter and η is the viscosity. The Henry function f(k.a) accounts for electrophoretic mobility changes induced via varying electrolyte concentrations in the solutions As a result of varying charge-to-size ratios (q/R) of different analytes, spatial separation and detection is possible, for example using a detector 108 of any appropriate type. Hence the charge to size/mass ratio of the various components can be determined for a calibrated system which gives valuable information regarding the composition of the analyte. However it is not possible to apply known methods in order to determine the molecular size/mass via capillary zone electrophoresis without knowledge of charge, as the size/mass and charge value are coupled to one another.

A further problem with known separation processes is that a permanently stored sample of the separated analyte is not available, meaning that analysis at a later stage is not possible. The invention is set out in the claims. According to a first aspect, it has been found, contrary to classical theory, that electrophoretic mobility is dependent on electric field. It has been found herein that electrophoretic mobility has a monotonic relationship with electric field. More particularly, it has been found that electrophoretic mobility has a linear relationship with the squared strength of the applied electric field. Furthermore it has been found that there is a well ordered relationship between field dependent mobility and mass and that this relationship is monotonic over a significant range. Thus, by measuring a plurality of values for electrophoretic mobility for plurality of field strengths, it is possible to decouple charge and mass from one another and obtain values for each in turn, allowing information regarding the analyte and its components to be determined. According to a second aspect, by depositing separated analyte on to a moving stage, an analyte track is created which can be stored and reused for future analysis. Embodiments will now be described, by way of example, with reference to the drawings of which:

Fig. 1 is a schematic diagram showing a known capillary zone electrophoresis apparatus;

Fig. 2 is a schematic diagram showing an electrophoresis apparatus according to a first aspect of the present invention;

Fig. 3a is a graph of linear coefficient of the total electrophoretic mobility (Y axis) against molecular weight (X axis) showing a generally inverse relationship between the first order coefficient and molecular weight;

Fig. 3b also shows linear coefficient of the total electrophoretic mobility against molecular weight, recorded for seven proteins in 20 mM sodium citrate pH3;

Fig. 4a is a is graph of first order coefficient of the total electrophoretic mobility (Y axis) against hydrodynamic radius (X axis) showing a generally inverse relationship between the linear coefficient and hydrodynamic radius;

Fig. 4b also shows the first order coefficient of the total electrophoretic mobility against hydrodynamic radius wherein the hydrodynamic radii have been measured via dynamic light scattering (DLS) in 20 mM sodium citrate pH3;

Fig. 5a is a graph of electrophoretic mobility (Y axis) against electric field (X axis) for Trypsinogen showing an approximately linear field dependence;

Fig. 5b shows the relationship between electrophoretic mobility and squared electric field strength for a variety of analytes at different concentrations, showing a generally linear relationship between electrophoretic mobility and squared electric field strength;

Fig. 6a is a schematic diagram of an apparatus according to a second aspect of the invention;

Fig. 6b shows schematically a deposited analyte according to the second aspect;

Fig 6c shows an optical read-out obtained from the deposited track;

Fig. 6d shows a 2DIR image and an electropherogram of a 3 protein mix deposited as a track;

Fig. 7 is a photograph of an analyte sample record created using the apparatus of Fig. 6;

Fig. 8 is a schematic diagram showing detail of the arrangement of Fig. 6a;

Fig. 9 is a diagram showing a two stage process according to the second aspect of the invention;

Fig. 10 is an electropherogram of a protein mixture compared with the equivalent protein track written onto a substrate and measured with intrinsic protein fluorescence.

In overview, and referring to Fig. 2, an arrangement according to a first aspect is shown. It will be seen that in addition to a first detector 108, further detectors 110, 112 are provided spatially separated from one another along the length of the capillary 104 - all typically connected to a computer or processor (not shown). This allows analyte parameters to be determined by measuring a plurality of values for electrophoretic mobility of an analyte or sample for a plurality of electric field strengths. Other arrangements are possible which include fewer detectors, for example just a single detector. The electric field dependence of the electrophoretic mobility of the analyte reported here, which until now has not been known, can be exploited to obtain molecular size and charge as well as other parameters such as diffusion coefficient, surface charge density and zeta potential of the analyte. For example this can be used for proteins to obtain protein size and charge and hence identify unknown proteins as part of a proteomics strategy. Using the detectors 108, 110, 1 12 the mobility for varying electric fields can be determined. Knowing the mobility can contribute to protein identification when other properties such as fragment sequence or amino acid content are also known by relying on, for example, mass spectrometry or optical identification with 2D infrared detection. As described in more detail below, the mobility of the analyte can be determined for varying electrical fields by increasing the electric field and detecting the corresponding analyte or analyte components at each of the detectors 1 12, 1 10 and 108 to determine the change in mobility against variations in electric fields It is possible to facilitate determination of the mobility of the analyte using a multi channel device such as a chip. A multi channel device can run multiple electric fields for one analyte at the same time or can run multiple analytes simultaneously at one or multiple different electric fields.

As mentioned above, the present applicant has found that there is a monotonic relationship between electrophoretic mobility and applied electric field. In particular it has been found that electrophoretic mobility varies linearly with the squared value of applied electric field strength. Thus, once the change in mobility against variation in electric field has been experimentally determined, and using a linear relationship between mobility and squared electric field strength, the coefficients of this linear relationship (gradient and intercept) can be determined. As described in more detail below, various parameters including mass can hence be derived from the determined coefficients. One such parameter which can be determined from mobility information is the molecular size, for example hydrodynamic radius R. Because, according to the presently-described methods, molecular size can be determined without prior knowledge of the electrostatic charge of the molecular species, the classical electrophoretic defining parameter - the charge to size ratio - is decoupled. As result, the molecular charge of the analyte system in the physicochemical environment (electrolyte buffer system) used for the investigation can be inferred, rather than being measured or calculated directly. Knowledge of molecule charge and related parameters can be critical in identification and classification of an analyte system. As discussed in more detail below, a simple calibration phase can be used to obtain coefficient values for the relationship between mobility and electric field strength, which then does not require repetition for a given system.

The simplest way of extracting the first order coefficient in the relationship is by injecting a sample at least twice, in separate experiments, at different field strengths and plotting the obtained mobilities against the squared electric field. For each injection, generally less than 0.5 nL could well be sufficient depending on the analyte concentration and detector sensitivity. In overview of a second aspect of the method described herein, analyte information can be recorded by combining a separation strategy, such as a mobility separation process on an analyte, with an optical readout. This enables the characterisation and identification of analytes such as intact proteins and other metabolites from complex mixtures. By depositing the separated analyte onto a moving stage to create an analyte track, intact molecular species can be created allowing further downstream experiments such as biochemical analyses upon recovery off the stage or sample plate. Samples can be presented to the spectrometer as dried tracks, that is thin films of material in which each discrete analyte component or protein can occupy an area well matched with the size of the laser beams being used in an optical readout system allowing an improved proteomics approach.

In the first aspect described above, the apparatus used can be a conventional capillary electrophoresis system with a capillary of any appropriate length, for example 10- 100 cm, sample plugs of volume, for example, 0.1-100 nL, a light source of wavelength, for example, 190-600 nm and voltage source of, for example, ± 10-30 kV for a typical capillary or corresponding electric field strengths when shorter capillaries or channels in microfluidic chips are used, and which can also be computer controlled to obtain the E-field variation. Experiments are run in typical capillary zone electrophoresis mode, with the capillary being immersed in an electrolyte buffer system which can be aqueous or nonaqueous, and analytes are introduced either hydrodynamically or electrokinetically.

Analyte parameters can be derived using the apparatus described above in light of the presently-described finding that the electrophoretic mobility of proteins and small molecules is monotonic with the squared strength of the applied electric field. The linear coefficient of the relationship - which can be assumed to be dominant, hence making the relationship approximately linear - is shown to be inversely dependent on the known molecular weight as well as on the hydrodynamic radius, as detailed below. In particular it is found that, although other facets may affect the relationship between electrophoretic mobility and applied electric field strength, the mass/size of the molecule under study is dominant. This is in contrast to current electrokinetic theory which does not recognise any electric field dependency of electrophoretic mobility, as can be seen from equations Ia and Ib above. In fact, the present applicant has shown for proteins and other analytes including small molecules that the electrophoretic mobility μ can be expressed in terms of the unmodified Einstein Stokes mobility value μ₀ given by equation (2) below together with a term proportional squared to the electric field E, as shown in equation 3 (below).

(2) Mo = 6πηR

(3) μ = γ E² + μ_o

As shown in equation (2), for a given analyte, the Einstein relation for the electrophoretic mobility μ₀ is a term inversely dependent on the hydrodynamic radius R of the analyte component and directly proportional to the charge q. A linear relationship between mobility and electric field as reported herein is thus represented by equation (3) having a proportionality coefficient γ effectively giving the gradient of the line and an intercept coefficient μo expressed in terms of the charge to size ratio and given by the Einstein relation.

Furthermore, referring to equation (4), it has been shown that the coefficient 7 exhibits an inverse dependence on the size of the analyte under investigation. That is:

(4)

γ oc—

R

More generally, the form is found to be (4') γ = a - R -b Hence it has been found that the proportionality coefficient 7 in the relationship between mobility and squared electric field strength is itself proportional to the inverse of the molecular size of the analyte under investigation. This relationship can generally be seen with reference to Figs. 3a to 4b which show the molecular weight and hydrodynamic radius varying inversely with the first order coefficient γ.

Application of this innovative experimental recognition allows more parameter information to be obtained than would be available according to classical electrokinetic theory, which recognises no relationship between electrophoretic mobility and the applied electric field. The Henry function f(k.a) attempts to numerically bridge observations of mobilities at low and high salt limits, respectively in classical theory. The intermediate regime of the Henry function arguments is subject of the investigation here. Referring once again to equation (3), it will be seen that varying the electric fields applied to an analyte and monitoring resulting mobility provides a generally monotonic relationship as can be seen from the graph of mobility (Y axis) against field (X axis) for trypsinogen in Figs. 5a and 5b. From the related coefficients of the linear relationship, additional data can be derived.

Hence if the system is calibrated once with an analyte of known parameters to obtain the constant value of the Henry-modified Einstein relationship (equation Ib) and to obtain the proportionally coefficient γ (equation 4), the gradient derived for an analyte or analyte component will yield a value for R and hence J the molecular size. From this, the mass can additionally be easily determined assuming a constant or known density for the analyte, for example the expected protein density, or may be obtained via extrapolation. Radius information also allows the value of charge, q, to be obtained separately from the Y intercept as 5 the value of R is known and the other values are known or calibrated.

It will be seen that the manner in which the electric field is varied can be selected from various possibilities. In a first approach, the mobility can be measured by a single detector in multiple runs each at different voltage or

10 electric field values. Yet further, the field or voltage can be varied during a single iun in which case a minimum of two detectors and preferably three detectors as shown in Fig. 2 allow the corresponding values of mobility to be determined. According to yet another approach, the field can be reversed and the mobility can be determined for each of the forward and reverse runs. A

15 minimum of two data points are required to allow determination of the coefficients in the linear relationship between mobility and squared electric field strength. But of course additional data points can be achieved allowing a better fit and enhanced accuracy.

20 Once the values of q and R have been determined as described above, additional parameter data can be obtained as required. For example, many proteins are characterised based on capillary zone electrophoresis experiments from the isoelectric point pi and databases mapping pi values to known analytes are available. The isoelectric point pi comprises the field value at

25 which the analyte becomes stationary and can be derived from the known q value of an analyte. Of course the analyte could be identified simply by its q value. Alternatively the isoelectric point, which is well characterised and is a common identifier, can easily be obtained and used in place of the q value, which is useful especially for proteomics applications. By way of example, protein Diffusivity D_p can be obtained from the known relationship: kj

(5) ^D _P = 6πηR

Where R is molecular size, k_B is the Boltzmann constant and T is the buffer temperature.

In addition, surface charge density σ$ can be obtained according to equation 6:

(6) σ_s ~ q/4πR²

The Deb ye length X₀ can be obtained from equation 7:

ε - D

(7) λ_D ^ι =

Where Dei is the counter ion diffusivity, σ is the conductivity of the solvent medium or solvent system and e is the dielectric constant of the solvent system.

The zeta potential Z or rather voltage drop in the Debye layer; can be further obtained from equation 8:

(8) Z = σ_s - λ_D lε . As a result, it will be seen that quick and uncomplicated molecular size determination is available via capillary zone electrophoresis or other field based mobility separation techniques, as a result of the relationship between mobility and electric field strength, described herein. The extraction of the molecular charge allows isoelectric point determination hence providing significant enhancement in protein characterisation and identification.

It is not necessary to include additional parts or components in conventional experimental setups other than potentially additional detectors and the approach can be employed as part of a separation strategy, for example with a pre-separation step of the kind shown in Fig. 9 such as chromatography or other. Any appropriate detector or process can be used including UV absorption, laser induced fluorescence, IR, THz or microwave The first aspect described above hence allows additional information to be obtained using the observed linear dependency between mobility and squared electric field in an electrophoresis set up, hence allowing decoupling of the mass and charge which was not previously possible using classical theory. Although the discussion of the first aspect above is principally in relation to proteins, it will further be noted that structurally similar or similarly sized analytes can equally well be analysed in this manner, for example small molecules, polymers, dendrimer systems, micellar and other lipid systems, DNA and other molecular systems. According to a second aspect, which can be used in combination with the first aspect or separately, a method and apparatus are provided for recording analyte information where the analyte is separated into components by a mobility separation process. The second aspect described herein can be understood with reference to Figs 6a, 6b and 6c. Referring firstly to Fig 6a, an analyte is subjected to electrophoresis in a vertical fused silica capillary 600 under an applied voltage 602. Analyte fractions or components shown generally at 604 move in a downward direction 606 in the capillary 600. The analyte is deposited via a buffer liquid cushion 608 onto a moving, conducting substrate plate 610, or dielectric plate with conducting plate underneath or a dielectric (polymeric) substrate with a conducting material patterned on top of it, moving in a direction 612 generally horizontal/perpendicular to the orientation of the capillary tube 600. Referring to Fig. 6b the deposition process results in a fractional pattern on the substrate plate shown by marks or deposits 616. It will noted that these are laid, in Fig. 6b, on a straight, tortuous or serpentine paths where the conducting plate 610 is movable in both the x and y directions in order to make more use of the area of the plate. A z axis is required to adjust the capillary tip to the substrate plate in order to create a sufficiently sized liquid cushion between capillary tip and plate. As described in more detail below, the deposited analyte can then be stored or subjected to optical readout for example using a two dimensional infra-red spectroscopy (2DIR) technique for various applications such as protein fingerprinting. The resulting read-out can be seen in the arrangement of Fig. 6c.

Referring to Fig. 7, a photograph showing deposition from the capillary tube 600 is shown alongside the corresponding track 614. Using the approach according to this second aspect, micrometer wide thin films can be prepared via an electro-coating process directly from capillary electrophoretic methods. The deposition of the analyte solution onto a conducting or dielectric substrate plate can be tightly directed via software controlled motion actuators (in one or two dimensions) beneath the substrate plate and a video microscope attached next to the depositing capillary. The motion actuators can be moved with a precision between 1 to 500 micrometers/minute and the video microscope resolves a 10 micrometer distance between capillary and substrate plate which enables determination of deposition conditions very accurately. The video microscope enables fine adjustment of liquid cushion size between capillary tip and plate in order to control substrate wetting to match size requirements for track widths.

As can be seen from Fig. 8, by controlling the relative speeds of deposition from the capillary 610 against movement of the slide or stage 614, relatively long, spaced analyte fractions or components 604 are deposited as relatively short, more closely spaced marks 616. Use of a buffer liquid cushion assists in the deposition process wherein, through the cushion, electroconnection is established and the analyte transport to the plate facilitated. The liquid cushion between the capillary tip and substrate is not a static but a dynamic entity, and hence will form part of the thin films eventually. Hence the deposited thin films not only contain the required analyte but also the electrolyte of the buffer system. Usually the electrolyte concentration will be much larger than the analyte and therefore the analyte could be considered trapped in a salt cage or salty gel if complete water evaporation can be prevented.

The composition of the eluting or dissolving fluid is irrelevant for the deposition process itself, since the desired depositing compounds are dissolved in the solvent and deposition occurs largely via electrostatic attraction between the plate (electrode) and the dissolved analytes. Volatile solvents are as suitable as water and for certain applications may be more useful. The approach extends to any electrolyte systems using pure solvents for the analyte. Pure solvents include any of: water, ethanol, methanol, acetonitrile, alkanes, and mixtures thereof; and ionic liquids and other organic solvents. Deposition of analytes returns a fractional pattern 616 that can be analysed via optical and spectroscopic methods in order to locate the fractions, on the one hand, and on the other to identify and quantify the constituents of the fraction. Any appropriate optical or spectroscopic methods can be adopted including 2DIR, Coherent Anti Raman Scattering (CARS), Raman scattering, fluorescence and so forth. Tight control of elution is required to make the optical read-out process efficient in terms of laser energy and analyte material being used. In particular, the process can be used as shown in Fig. 8 to match the deposited fraction dimension to the laser spot size, providing improved accuracy and efficiency.

In one approach a sample record can be generated using the following parameters:

• For electrodepositions at atmospheric pressure, employable applied field strengths vary from 0.01 to about 15 kV/m, depending on the conductivity of electrolyte system (can be non-electrolyte system to prepare thin film from solute);

• Capillary lengths can vary from about 0.5 to 200cm. Capillaries are heat-tapered to produce a capillary tip that can match the required track widths. For an original capillary width of: 360um (i.d. 50 um), heat- tapering enables conical tips widths between 1 -360 um and the diameter of the inner channels shrinks proportionately;

• Track writing (electrodeposition) can be pressure-assisted via siphoning

(height difference between capillary inlet and outlet) or via using a pump; • Substrate plates for track deposition can be, for example, polished stainless steel, Teflon, glass, or any polymeric or dielectric substrate that is sufficiently thin and has a conducting substrate connected beneath it;

• Liquid flow rates usable are about 0.1- 100 nL/min to produce track widths that match laser beam diameter of about 30-300 microns;

• Electrolyte concentrations can be from 0 to 10OM;

• Substrates should ideally exhibit a low optical background for the measurement method to be used. e.g. fluorescence or 2DIR spectroscopy.

As will be seen, a generated sample record can be applied directly for an optical or other read-out in conjunction with the motion stage using, for example, a detector 618 as shown in Fig. 8. Alternatively, the sample record can be stored for future use as it dries very quickly. The slow deposition rates and slide movement thus allow a physical record of the actual analyte/analyte fractions rather than simply the data derived therefrom to be stored.

It will be appreciated that the second aspect described above can be used in relation to any appropriate analyte including, but not limited to, proteins and small molecules, polymers, DNA, protein, dendrimer systems, micelles and other lipid systems such as liposomes, and other more complex molecular systems such as viruses, bacteria and cells. It can be used for any appropriate concentration and can be used in conjunction with a prior stage such as a chromatography stage 620 as shown in Fig. 9 to separate the analyte. It also allows analysis, if desired, per chromatography track.

Figure 10 shows an electropherogram of a 3-protein mixture compared with the protein track being written onto substrate and measured with intrinsic protein fluorescence. The different relative peak sizes in the electropherogram reflect the fact that the electropherogram was created by monitoring protein absorption as the protein passed through the capillary whilst, on the hand, the track of the protein is read by means of protein fluorescence. This comparison demonstrates that the absorption and fluorescence properties of a protein are not simply related. The usefulness of protein tracks written onto a substrate for analysis of proteins, either alone or in conjunction with other analysis techniques such as electropherograms, is thus demonstrated.

Hence an arrangement is provided which allows separation of the constituents of a complex mixture via capillary electrophoretic separation methods and maintaining the achieved resolution onto a substrate plate. The separated fractional constituents can be stored separately under suitable conditions - this is assisted by the extremely slow motion of the motion stage or slide allowing the separated components to dry immediately. Hence any appropriate optical and spectroscopic read-out can be applied for fraction detection and identification either in real-time or by retrieving a stored slide. The nondestructive approach allows the constituents to be maintained intact and reused in further investigations such as biochemical reactions to test activity, and Nuclear Magnetic Resonance (NMR) analysis to investigate structures for mass spectrometry techniques such as MALDI aiding in analyte, in particular peptide or protein identification by elucidating amino acid sequences or atomic constitutions.

Yet further the approach allows preparation of high density molecular thin films of any shape that can be used for general structural, chemical and compositional analysis of materials and enables preparation of thin films under applied electric field conditions, for example, electrocoating. Thin films can be prepared onto a conducting substrate directly or onto a dielectric material and the thin film properties can be tightly controlled by adjusting solution composition and deposition conditions to allow flexible deposition and electro- coating parameters to be applied.

The arrangement provides particular advantages in the area of drug discovery in order to find additional information about where the drug binds to the protein, the strength of any such bindings and so forth. Similarly it can also be used to study protein-protein and protein-nucleic acids.

It is possible to wash an array of proteins, printed as described herein according to the second aspect, with a drug or drug candidate or metabolite or protein, peptide or nucleic acid and see where it binds using optical methods. One can then identify which proteins have captured the drug/metabolite or other binding molecule. As an alternative to washing the protein tracks, the drug/metabolite or other binding molecule can bind into the cell lysate or sample before separation. Some drug/small molecules or other binding molecules will remain attached even during the separation process and subsequent track writing, giving the same result of a drug screen. In a third variant the small molecule metabolites or drug/drug candidates or other binding molecules may be introduced into the running buffer to allow them to equilibrate with the proteins in that way. A range of methods in which capillary electrophoresis is used for the determination of binding constants is well known. For example, see the review by C. Jiang and D.W.Armstrong,(2010) Electrophoresis 31, p.17-27 and references therein. The key difference here is writing the proteins with their molecular binding partner(s) still bound onto a substrate as a track of protein such that they can be analysed as described in the present application.

This drug discovery mode of operation allows washing the tracks with small molecule binders then washing off and finding where they are bound, as well as pre-addition of small molecule binders for attaching to the protein, and introduction of small molecule binders to the running buffer to allow them to bind from there. This last approach can reduce the loss of pre -bound

molecules; they will tend to concentrate on those proteins that have good affinities and, as they are small, they can diffuse over a larger distance to deplete part of the buffer and concentrate where they bind.

Hence mass extraction methods can be used to distinguish between complex ED protein species to allow identification of a protein as a monomer or dimer. For example, consider an electrophoresis experiment as discussed previously. Injection can be performed using a sample containing two added (known) analyte species. The data analysis (electropherogram) however reveals three species. Now the mass/radius method using the calibration curve returns three first order coefficients, one for each species. Comparison of the coefficients with the calibration curve will confirm the known masses of the two purposely added analytes. The third coefficient should return a value corresponding to the sum of both added analyte species and hence confirms complexation of the two initially added analytes.

It will be noted that, in relation to the second aspect, separation of the analyte is discussed with reference to use of electrophoresis as the sole or final stage. However any mobility separation process can be adopted as the final separation stage including chromatography.

It will be appreciated that the aspects described throughout the present application can be combined or juxtaposed with each other and/or with other process steps as appropriate. Any appropriate form of mobility separation technique including electro-osmosis can be applied to any appropriate sample or analyte, and subsequent analysis for read-out can be performed using any appropriate detection/analysis technique.

Claims

1. A method of determining an analyte parameter comprising measuring a plurality of values for electrophoretic mobility of the analyte for a plurality of electric field strengths, determining a coefficient of a relationship between mobility and applied electric field strength and deriving a parameter from the coefficient.

2. A method as claimed in claim 1 in which the relationship is linear, for example of the form y = ax + b.

3. A method as claimed in claim 2 in which an analyte dimension parameter is derived from the gradient, (a) coefficient.

4. A method as claimed in claim 2 or claim 3 in which an analyte charge parameter is derived from the intercept (b) coefficient

5. A method as claimed in any of claims 2 to 4 in which the linear relationship is given by the equation μ = γ - E² + μ₀ = γ - E² + _—J -_— where

6πηR γ oc— and more generally γ = a - R^~b , μ is the mobility, E is the electric field, R

q is the charge, R is the hydrodynamic radius and τ\ the dynamic viscosity.

6 A method as claimed in any preceding claim in which the plurality of electric field strengths are obtained by one of:

varying the electric field;

increasing the electric field;

decreasing the electric field; repeating the measurement for varying electric fields;

reversing the electric field.

7. A method as claimed in any preceding claim in which the analyte is one of a protein, small molecular, polymer, dendrimer system, micellar or other lipid system, DNA or other molecular system such as a virus, bacteria or cell

8. A method as claimed in any preceding claims in which the parameter is at least one of charge;

diffusion coefficient; surface charge density; size; radius diameter; volume; zeta potential (or isoelectric point).

9. A method as claimed in any preceding claim further comprising deriving additional information regarding coefficients of the linear relationship in the calibration phase.

10. A method of spectroscopy or proteomics comprising a method of any claims 1 to 9.

1 1. A method as claimed in any preceding claim in which electrophoretic mobility is obtained by measuring the time taken for an analyte or analyte component to traverse a distance between detection points.

12. An electrophoresis apparatus comprising means for varying an electric field strength in an electrophoresis mobility region and at least one detector for detecting mobility of an analyte for each of a plurality of electric field strengths.

13. An apparatus as claimed in claim 12 in which the detector comprises one of a UV absorption laser induced fluorescence, infrared, terahertz, or microwave detector.

14. A computer program arranged to control an electrophoresis apparatus to perform the method of any of claims 1 to 11.

15. A computer or computer readable medium storing therein instructions for implementing the computer program of claim 13.

16. A method of storing analyte comprising subjecting the analyte to a mobility separation process, and depositing the separated analyte onto a moving stage to create a separated analyte track.

17. A method as claimed in claim 16 in which the mobility separation process comprises one of electrophoresis, electroosmosis or chromatography.

18. A method as claimed in claim 16 or 17 further comprising subjecting the separated analyte track to an optical read-out.

19. A method as claimed in claim 18 in which the analyte track dimension is mapped to an optical read-out dimension, for example a laser spot dimension.

20. A method as claimed in any of claims 16 to 19 in which the moving stage comprises a slide or substrate plate.

21. A method as claimed in any of claims 17 to 20 further comprising storing the track.

22. A method of spectroscopy comprising a method of any of claims 17 to 20.

23. A method as claimed in claim 22 further comprising applying an optical read-out step to the moving stage.

24. A method as claimed in claim 22 further comprising storing the stage and separated analyte track for future analysis.

25. A method of electro-coating a substrate with an analyte comprising a method of and of claims 16 to 24.

26. An apparatus for creating a record of an analyte comprising a mobility separation component and a movable stage adjacent thereto, the mobility separation component being arranged to deposit separated analyte onto the moving stage to create a track of separated analytes.

27. An apparatus for analyzing an analyte comprising a mobility separation component, a moveable stage and an analyser in which the mobility separation component is arranged to deposit separated analyte onto moveable stage to create a separated analyte track and the analyser is arranged to perform an analysis step on the separated analyte track.

28. An apparatus as claimed in claim 27 in which the analyser comprises an optical read-out component.

29. An analyte record comprising a substrate having thereon a separated analyte track creating according to the method of any of claims 16 to 25.

30. A method of analysing a bindable substance comprising storing an analyte as claimed in any of claims 16 to 25, the analyte comprising a binding analyte and applying the bindable substance to be binding analyte.

31. The method as claimed in claim 30 in which the bindable substance comprises one of a drug, drug candidate or metabolite.

32. The method as claimed in claim 30 or claim 31 in which the binding analyte comprises a protein or a plurality of proteins.

33. The method as claimed in any of claims 30 to 32 in which the bindable substance is applied to the binding analyte by one of washing a separated analyte track, introducing the bindable analyte prior to the mobility separation process or during the separation process.

34. A method of distinguishing between complexed protein species comprising applying the protein species in the method of any of claims 30 to 33.

35. A method or apparatus substantially as described herein with reference to the drawings.