A method of analyzing a first compound of a fluid sample and an apparatus therefor

Abstract

The invention relates to a method for analyzing a first component of a fluid sample also comprising a second component where the sample is stepwise introduced further and further along a substrate and finally removed from at least the last wetted portion of the substrate. The analysis is performed at the last wetted portion of the substrate.

Description

[0001] A METHOD OF ANALYZING A FIRST COMPOUND OF A FLUID SAMPLE AND AN APPARATUS

[0002] THEREFOR

[0003] The present invention relates to a method of analyzing a first compound in a fluid sample and especially for analyzing the first compound in the sample also comprising a second compound.

[0004] Relevant technology may be seen in WO17 / 191080, EP 0211334, “Centrifugally enhanced paper microfluidics”, Neus Godino, et al., 2012-01-29; “Development of a simple device for processing whole blood samples into measure aliquots of plasma”, Carl Burtis, et al., 1986- 09-01, EP2878373, US2016 / 047794, WO02 / 43866, JP2009 / 014529, “A centrifuge-pneumatic cascade for fully integrated and multiplexed biological analysis”, Neus Godino, et al., 2012- 01-29, US2015093771 , WO0243866, "Paper on a disk: balancing the capillary-driven flow with a centrifugal force." Lab on a Chip 11.20 (2011): 3404-3406 by Hwang, Hyundoo, et al., EP0211334, US2015093771 , WO02 / 43866, and Lab on a Chip 11.20 (2011): 3404-3406.

[0005] In a first aspect, the invention relates to a method of analyzing a first component of a fluid sample comprising the first component and a second component, the method comprising: providing a rotatable element having an axis of rotation comprising: o a sample chamber comprising a sample inlet positioned farther away from the axis of rotation than a portion of the sample chamber, o a substrate provided at least partly in the portion of the sample chamber, o a sample receptacle positioned closer to the axis of rotation than the sample inlet and o a sample feed line from the sample receptacle to the sample inlet providing the sample in the sample receptacle, feeding sample into the sample chamber by, in each step of a sequence of steps, increasing a rotation frequency of the rotatable element, removing sample from at least a first portion of the substrate having received sample during the last step of the sequence of steps, and perform ing a quantitative analysis of the first component in the first portion, where: each step of the sequence of steps comprises increasing the rotation frequency from a first rotation frequency to a second rotation frequency within no more than 1 s and with a rotation frequency increase of at least 1 Hz.

[0006] I n this context, the analysis of the first component m ay be any type of analysis. The analysis may be supported by or include the presence of the first com ponent on the substrate. The analysis m ay be perform ed while the substrate is in the sam ple chamber, or the substrate may be removed from the sample chamber before the analysis is perform ed.

[0007] The analysis may be quantitative such as to determ ine a concentration of the first com ponent, such as in the first portion and / or in or on a predeterm ined area or volum e of the first portion of the substrate. Alternatively, the analysis may be to determ ine e.g. a presence or a m inim um concentration or amount of the first component.

[0008] The analysis may be electrochem ical, optical, electrical, magnetic, piezoelectric, spectroscopic or the like.

[0009] Preferably, the analysis is based on an intensity of radiation or light em itted from or scattered by the first com ponent. It m ay be desired to collect such intensity from a portion only of the surface of the substrate, such as a portion which received the sample only during the last step of the sequence of steps. An averaging or the like may be m ade over the substrate and along lines perpendicular to the direction of flow of the fluid during the steps of the sequence of steps. I n this m anner, an output signal, such an spectrum or band may be obtained of such intensity or the like representing the intensity em itted from the first com ponent on the substrate at least for desired portions of the substrate. Other portions of the substrate m ay be used as e.g. a background so that any radiation received from such portions m ay be subtracted from that received in the selected portions. The intensity or the like m ay be received from a portion of the substrate such as a predeterm ined percentage of a length of the substrate along the direction of the flow of the sam ple. 1 -50% , such as 10- 30% , such as 15-25% of the length m ay be selected. Alternatively, the above curve m ay be used, and only the portion of the substrate in the FWHM or other m easures of the curve may be used. A m axim um of the intensity curve m ay be determ ined and only the portion around the top having a predeterm ined percentage of the m axim um may be used. The sample is fluid and may be a liquid. The sample comprises the first and second com ponents which m ay be molecules or another type of component. The sam ple m ay be a biological fluid, such as blood, serum , urine, m ilk, saliva, sweat, or the like. Alternatively, the sample m ay be a fluid from a production or test facility where the first com ponent is desired detected or analyzed.

[0010] The first com ponent m ay be any type of com ponent, such as a drug. The first component may not be usual in the type of sam ple. Thus, the sam ple may be blood or blood serum and the first com ponent m ay be a drug adm inistered to a patient from which the sam ple was drawn.

[0011] The first com ponent m ay be a relatively sm all molecule, such as within the interval of 3-7 kDa and / or any type of molecule which is SERS active, meaning that it has functional groups that could attach to the substrate surface, such as am ines, sulfur, or the like

[0012] The first com ponent m ay attach or adhere to the substrate so that a first force is required to remove a molecule or predeterm ined amount of the first component from a surface of the substrate. The adherence or attachm ent may be caused by any desired effect, such as an electrostatic attraction or attachment. Alternatively, or additionally, the first com ponent may be sticky and thus adhere to the substrate via its stickiness.

[0013] The second component m ay be any type of component, molecule or the like. The second com ponent may be a natural part of the sam ple, such as fat globules, protein, cells, or the like of a sam ple derived from a vertebrate. Naturally, the second com ponent m ay be any other com ponent from any other type of sample, such as biological fluids, cells or vesicles.

[0014] The second component m ay attach or adhere to the substrate so that a second force is required to remove a molecule or predeterm ined amount of the second component from a surface of the substrate. Again, the adherence or attachm ent m ay be caused by any desired effect, such as an electrostatic attraction or attachment. Alternatively, or additionally, the second com ponent may be sticky and thus adhere to the substrate via its stickiness. I n a particular situation, the substrate may be configured to physically trap or block portions of the second component of the sam ple from moving relative to the substrate. This is explained further below.

[0015] For the sam e number of molecules or the like, or for the sam e amount of the second com ponent, the second force is larger than the first force, so that if a force is applied exceeding the first force but not the second force, a larger num ber or amount of the first com ponent will be dislodged or de-attached from the substrate. The hydrophobic or hydrophilic properties of the metallic surface can have an effect on the adherence of the components where a hydrophilic surface m ay have a higher adherence / stickiness to all of the components of a biological sample. Hydrophobic surfaces of e.g. the metallic pillars of the preferred substrate type, can be turned into hydrophilic surfaces by the adding to the sam ple an organic solvent, like ethanol or methanol, as this will favor the molecular m igration and adherence of com ponents in the sam ple.

[0016] I n this context, the rotatable elem ent is an element configured to be rotated around the axis of rotation. The rotatable element may com prise m eans, such as a cavity or hole / channel for receiving an axle of a rotating device m uch like a DVD. I n that situation, the axis would extend through the axle. Alternatively, the rotatable element may be configured to be supported on a surface while rotated.

[0017] The rotatable element may com prise one or m ultiple sample chambers each com prising a substrate. Multiple sam ple receptacles and sample feed lines may be provided if different sample chambers are to receive different sam ples.

[0018] When the sample inlet is provided farther from the axis than a portion of, such as all of, the sample chamber, the rotation will provide a force acting on sample in the sam ple cham ber in a direction toward the sam ple inlet.

[0019] Then, a substrate provided at least partly in the portion of the sample chamber m ay be drained or substantially drained by allowing sample in the sample chamber to escape from the sam ple cham ber through the inlet. This evacuation may be obtained in any desired manner, such as by using a pump.

[0020] On the other hand, when the sam ple receptacle is positioned closer to the axis of rotation than the sample inlet and when a sample feed line is provided from the sam ple receptacle to the sam ple inlet, rotation may force sam ple from the sample receptacle to the sample inlet via the sam ple feed line.

[0021] Rotation may then be used for controlling the flow of sample into and out of the sample chamber.

[0022] The m ethod com prises, generally, providing the sam ple in the sam ple receptacle, feeding sample into the sam ple cham ber, removing sample from at least a first portion of the substrate having received sam ple during the last step of the sequence of steps, and perform ing the quantitative analysis of the first com ponent in the first portion. The analysis is performed in the first portion. Preferably, the analysis is perform ed in or at a portion of the substrate which was provided with sample in the last step or the last steps. Thus, the analysis m ay be perform ed on or at a portion of the substrate which received a portion of the sample during the last step, penultimate step or third-to-last step. An advantage of this is that sample m ay be more easily removed from this portion of the substrate. Another advantage is that a relatively high concentration of the first com ponent is seen there.

[0023] The sample m ay be provided into the sam ple receptacle in any desired manner. The sam ple receptacle m ay com prise an opening to outside of the rotatable elem ent so that the sam ple may be fed into the sam ple receptacle.

[0024] The feeding step comprises rotating the rotatable elem ent to generate the sample feeding. According to the invention, the feeding is provided in a sequence of steps where each step com prises increasing a rotation frequency of the rotatable element. Each step comprises increasing the rotation frequency from a first rotation frequency to a second rotation frequency, where the first and second rotation frequencies are different for each step. For each step, the first and second rotation frequencies increase compared to the form er step.

[0025] According to this aspect of the invention, each step of the sequence of steps comprises increasing the rotation frequency from a first rotation frequency to a second rotation frequency within no more than 1 s, such as within no more than 0.7s, such as within no more than 0.5s, and with a rotation frequency increase of at least 1 Hz, such as at least 2Hz, such as at least 3Hz, such as at least 4Hz, such as at least 5Hz.

[0026] I n this context, an increased rotation frequency m eans that an increased amount of sample is present in the sample chamber. Thus, each step results in an in-flow of sample into the sam ple chamber. This causes the sample to flow vis-a-vis the substrate surface and will thus dislodge first and second component portions and move these with the flow of sample.

[0027] Preferably the first rotation frequency of one step is equal to or higher than the second rotation frequency from a neighboring and previous step. I n this manner, no decrease of rotation frequency takes place during any of the steps.

[0028] Any number of steps may be performed, such as 2, 3, 4, 5, 6, 7, 8, 9, or more, such as 2- 100, such as 3-50, such as 4-20 steps.

[0029] According to the invention, the rotation frequency increase of each step is sufficiently abrupt to create a forceful flow in the sample chamber and relative to the surface of the substrate. This forceful flow will act to have the sam ple cover, for each step, more and more of the surface of the substrate, in a direction toward the axis of rotation. At the sam e time, the forceful flow will act to dislodge at least a portion of the first com ponent already present in the sam ple cham ber and attached (or otherwise attracted to or engaging the substrate) to the substrate, so that the dislodged portion or the first component will follow the flow in the direction of the rotation axis.

[0030] On the other hand, it m ay be desired that the rotation frequency increase of each step does not result in a flow large enough to dislodge a predeterm ined proportion of the second com ponent so that, during the steps, the second com ponent will tend to attach to the substrate at the first wetted portions (the portions receiving sample in the first step(s)) of the substrate whereas the first com ponent will to a larger degree move with the sam ple toward the axis of rotation and thus the last (or latter) wetted portions of the substrate.

[0031] Preferably, the acceleration of the rotatable elem ent takes place, in each step, during 0- 1 s, such as 0.25-0.75s, such as around 0.5s. Then, the positive, acceleration of the sam ple vis- a-vis the substrate may take place during 0-2s, such as during 0.5- 1.5s depending on parameters of the sample and the flow channel. The positive acceleration can be followed by a negative acceleration as the relative sample movem ent will slow down vis-a-vis the substrate, to arrive at, if the rotatable elem ent rem ains at that rpm , new extent over the substrate in the direction toward the axis of rotation.

[0032] It is preferred that a step causes the sam ple to extend 0.1 -10m m, such as 0.2-0.6m m , such as 0.3-0.5m m farther in the direction of the axis of rotation.

[0033] Thus, the m axim um acceleration for each step, of the sam ple vis-a-vis the substrate may be on the order of 0.1 -10mm / s2, such as 0.5-5m m / s2. This acceleration may be seen within the during 0-2s, such as within 0.5- 1 .5s of the time duration of a step. Outside this period of time, a lower acceleration, or no acceleration, may be seen. I n this context, the acceleration preferably is along the circular movem ent of the sam ple.

[0034] For example, if the sam ple inlet is provided 4cm from the axis of rotation, the increase in rotational frequency of the rotating element may be 1 - 100Hz / s, such as 2-50Hz / s, such as 5- 25Hz / s, such as 7- 15Hz / s and each step may increase the rotation frequency by 1 -50Hz, such as 2-25Hz, such as 3-20Hz, such as 4- 10Hz. As the centrifugal force correlates with the distance to the axis of rotation (radius) and the cube of the angular velocity, twice the distance would generate twice the force, and twice the angular velocity would generate 8 tim es the force. Thus, the present rotation frequency can easily, for e.g. the 4cm radius, be converted into other rotation frequencies and / or radiuses to arrive at the same overall force and acceleration.

[0035] Naturally, if the distance from the sample inlet to the axis of rotation changes, the rotation frequencies and thus the accelerations may be adapted correspondingly.

[0036] It may be desired to provide a delay or pause between the steps or at the rotation frequency increases of the steps. This delay or pause may allow the first and / or second components to attach or re-attach to the substate between the accelerations. Such pauses may be 1 - 100s, such as 2-50s, such as 3-20s, such as 4-8s, such as 4-6s. The delays may on the one hand be selected large enough to allow the (re)attachment and on the other hand not prolong the analysis time duration excessively.

[0037] I n one em bodiment: the feeding step com prises, in each step of the sequence of steps, increasing a gas pressure inside the sam ple cham ber and the removing step com prises the increased gas pressure forcing the at least portion of the sample out of the sam ple cham ber.

[0038] Operation of this type m ay be seen in W017 / 191080, which is hereby incorporated by reference, where the sample cham ber has a single opening through which the sam ple is introduced. When only that opening is provided and the sam ple is introduced therethrough, and when it is provided at the far end of the sample cham ber, no air (to any substantial degree) can escape the sam ple cham ber. Then, the higher the rotation frequency, the farther will the sam ple extend in the sample chamber in the direction of the axis of rotation and the higher will a gas pressure in the sam ple cham ber be.

[0039] I n that embodim ent, the removing step may com prise reducing a rotation frequency of the rotatable elem ent. Decreasing the rotation frequency will then allow the high gas pressure to force at least part of the sam ple out of the sam ple cham ber, where the sample will then initially be forced away from the last wetted portion of the substrate. It m ay be desired that the decrease in rotation frequency is not as abrupt as the rotation frequency increases of the steps, as this m ay cause excessive dislodging of the first component from the substrate back into the sam ple and thus transport thereof out of the sam ple com partment.

[0040] The removing step may comprise a smooth or constant reduction of the rotation frequency. Alternatively, the rotation frequency m ay be reduced also in steps. The steps may be as those of the acceleration process where noticeable decelerations are provided in individual steps, where the acceleration is now a deceleration. The RPM reductions may be as the RPM increases, the time periods of the reductions may be as the increases and the waiting times may be the same. Alternatively, it m ay be desired to decelerate with a lower RPM reduction per step or a larger tim e for a given RPM reduction so that the decelarations are more gentle.

[0041] I n addition, or alternatively, the removing step may com prise providing a suction force acting from the sam ple cham ber and towards a waste chamber. The suction force may be caused by capillary forces as described further below. This suction force may em ulate the removal operation described above and below - i.e. a stepped removal operation or a more smooth removal operation.

[0042] I n general, it m ay be desired to allow a period of time between the last acceleration step and any removal step, such as a reduction in rotation frequency or providing a suction force. This period of tim e may be 5-200s, such as 10- 100s, such as 15-50s if desired. This may allow the analyte to adhere or attach better to the substrate before the sam ple is removed.

[0043] Substrates for e.g. optical analysis may be trend m aterials or structures based on metals, oxides (silica, SiO2, TiO2) and chem ically fabricated nanoparticles (polystyrene, other polym ers) . It may be desired that the substrate has a m inim um surface roughness not only due to the analysis but also as this increases the adherence to certain types of second com ponent, such as protein.

[0044] I n a preferred embodim ent, the substrate comprises a base with pillars extending therefrom , the pillars being spatially displaced from each other. The pillars m ay have a first end attached to the base and a second end. The pillars may be parallel to each other or may be bendable and be able to lean to each other. The pillars may be made of silicon and / or fused silica and may have an electrically conducting surface on the second end. I n one situation, a pillar, or preferably more than 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95% of the pillars, preferably has / have a height being at least 2 times a mean spacing between the pillars. Preferably, the pillars may have a height, i.e. the distance between the base and the second end, of 20- 1000nm , such as 30-500nm , such as 50-400nm , such as 75-300 nm . One, more or all pillars may have a height of at least 30nm , such as at least 40nm , preferably at least 50nm , such as at least 100nm , preferably at least 200 nm , such as at least 400, 500, 600, 700, 1000, 1200, 1400 or 1600 nm . The substrate of this kind m ay be configured for Surface Enhanced Raman Scattering (SERS) applications. The second end m ay comprise a tip m ade of metal such as gold, silver, alum inium , copper, platinum , or a sem iconductor material, such as one from a group of I I I V sem iconductors. The tip is a surface at least substantially covering a part of the pillar, which part is the farthest from the base along the pillar. The tip needs not to cover any part of the pillar between the second end and the base.

[0045] Such pillars may be used for enhancing Ram an Scattering to facilitate a SERS type measurem ent on the first com ponent thereof.

[0046] Thus, firstly, the material of the pillars as well as the spacing there between m ay be tailored to the first component.

[0047] I n addition, and especially in situations where the first com ponent is sm aller in size than the second com ponent, such as if the first com ponent is a relatively small molecule and the second com ponent is protein, it is preferred that the sam ple cham ber has an internal height, at least at positions at which the substrate exists, of no more than 10 tim es a height of the pillars, such as no more than 5 tim es, 4 tim es, 3 times or 2 times the height of the pillars, above the base of the substrate. I n addition, an internal width, perpendicular to a direction toward the axis of rotation, of the sam ple chamber, at the substrate, may be desired no more than twice, such as no more than 1.5 tim es, such as no more than 1.25 times, such as no more than 1.1 times, an extent of the substrate along the sam e direction. I n this manner, a relatively large proportion of the sample, travelling in the sam ple chamber toward the axis of rotation during the steps, will travel between the pillars. I n this m anner, physical trapping of the second component m ay become a factor in the retaining of the second com ponent while the first component, during the steps, moves with the sample in the direction toward the axis of rotation.

[0048] As m entioned, the feeding step preferably comprises feeding sample into the sam ple container in each feeding step. Then, an increasing amount of sample is added to the sample container during the sequence of steps. This may be obtained when the sample feed line allows sam ple flow during all feeding steps. The sam ple feed line m ay not be blocked at any point in time. A blocking could be a design which allows fluid flow only at a predeterm ined m inim um rotation frequency. Preferably, the sam ple feed line allows fluid flow even at the rotation frequency of the first step in the sequence of steps in the feeding step.

[0049] Another aspect of the invention relates to an assembly com prising a rotating device, an analyzer, and a rotatable element having an axis of rotation, wherein the rotatable elem ent com prises: a sam ple cham ber com prising a sample inlet positioned farther away from the axis of rotation than a portion of the sample chamber, a substrate provided at least partly in the portion of the sample chamber, a sam ple receptacle positioned closer to the axis of rotation than the sam ple inlet and a sam ple feed line from the sample receptacle to the sample inlet and wherein the rotating device is configured to rotate the rotatable elem ent around the predeterm ined axis to: feed sam ple from the sample inlet to the sample chamber by, in each step of a sequence of steps, increasing a rotation frequency of the rotatable elem ent, each step of the sequence of steps com prising increasing the rotation frequency from a first rotation frequency to a second rotation frequency within no more than 1 s and with a rotation frequency increase of at least 1 Hz and remove sam ple from at least a first portion of the substrate having received sam ple during the last step of the sequence of steps, and wherein the analyzer is configured to perform a quantitative analysis of the first component in the first portion.

[0050] Generally, all em bodim ents, situations and considerations m ade in relation to the first aspect of the invention m ay be equally relevant in relation to all other aspects of the inventions.

[0051] Thus, the rotatable element m ay be as described in relation to the first aspect of the invention, as m ay the substrate, the sample, the first com ponent and the second com ponent.

[0052] The rotating device may be configured to receive the rotating elem ent or at least engage this in order to rotate the rotatable elem ent around the axis of rotation. The rotating elem ent may comprise an axle or the like for engaging the rotating element, such as extending through a potential opening or channel in the rotatable elem ent. The rotating device is capable of controlling the rotational frequency of the rotatable element. The rotating device thus may comprise a motor or drive, gears, transm ission or the like, which m ay be controlled in order to arrive at the desired rotation of the rotatable elem ent.

[0053] Naturally, the analyzer m ay be for optical analysis, where the analyzer has a source of radiation and a radiation receiver, where the wavelengths etc. are adapted to the analysis to be performed. The analyzer m ay be configured to receive the rotating elem ent and / or the substrate to perform the analysis. The analyzer m ay be attached to the rotating device or even the rotatable element if desired. The rotating device would be configured to generate the rotation, rotation frequencies, accelerations and delays described above.

[0054] As described above, a preferred substrate com prises a base with pillars extending therefrom , the pillars being spatially displaced from each other, the pillars having a first end attached to the base and a second end.

[0055] As also described above, especially or substrates of this type, the sample chamber has internal dim ensions adapted to the dim ensions of the substrate. Thus, it is preferred that the sample chamber has an internal height, at least at positions at which the substrate exists, of no more than 10 tim es a height of the pillars, such as no more than 5 tim es, 4 tim es, 3 tim es or 2 times the height of the pillars, above the base of the substrate. I n addition, an internal width, perpendicular to a direction toward the axis of rotation, of the sample chamber, at the substrate, may be desired no more than twice, such as no more than 1.5 tim es, such as no more than 1.25 tim es, such as no more than 1.1 tim es, an extent of the substrate along the sam e direction. I n this m anner, a relatively large proportion of the sam ple, travelling in the sam ple cham ber toward the axis of rotation during the steps, will travel between the pillars. I n this m anner, physical trapping of the second com ponent may becom e a factor in the retaining of the second component while the first com ponent, during the steps, moves with the sam ple in the direction toward the axis of rotation.

[0056] I n the above situation, the delivery of the sample on to the substrate is described as performed by rotating rotatable elem ent having the substrate and the sample, so that the sam ple is delivered in waves or bursts farther and farther along the surface of the substrate. Naturally, the same effect m ay be obtained using other manners of sam ple delivery, such as by providing the substrate in a stationary sam ple cham ber where on or more pumps or the like are used for the introduction of the sam ple into the sample chamber and over the substrate surface in the same manner. A further alternative would be dipping the substrate, even without the sample chamber, in the sam ple, where the dipping speed and acceleration defines the forces applied to the first component and the second com ponent.

[0057] Also, the removal of the sam ple over at least the last wetted portion may be obtained in a number of m anners. If a stationary sample chamber is used with pum ps for delivering the sam ple thereto, the sam e or other pump(s) m ay be used for removing the sam ple from at least the last wetted portion. It is noted that in the situation where the sam ple cham ber has only one opening, the built-up gas pressure therein may by itself force sample out of the cham ber. No rotation is required as the gas pressure itself may be sufficient for at least removing the sample from the portion of the substrate at which the analysis is desired. Thus, an aspect of the invention relates to the overall movem ent of the sam ple over the surface of the substrate irrespective of the m eans provided for the transport of the sample over the substrate as well as the means provided for evacuating the at least portion of the substrate.

[0058] I n general, this aspect relates to a method for analyzing a sam ple on a substrate, the m ethod com prising applying a sam ple to the substrate, the sample being a liquid com prising a first com ponent and a second component, wherein the applying step com prises supplying the sam ple to the substrate in a sequence of steps, wherein: in each step, the sam ple extends farther, in a predeterm ined direction, along the substrate surface, a delay of at least 1 s is provided between two neighbouring steps, and each step is performed so that at least part of the sample, during the movem ent in relation to the sample reception surface, experiences an acceleration of at least 0.1 m m / s2in the predeterm ined direction, and the method having a final step of removing at least a part of the sam ple from a first portion of the substrate surface and perform ing an analysis of the first portion of the sam ple reception surface.

[0059] Naturally, the accelerations mentioned above will be equally relevant for this more generic aspect of the invention.

[0060] Clearly, the substrate m ay be provided in the sam ple chamber as defined above, but even stepwise dipping of the substrate into the sample will be effective.

[0061] The substrate, sample, first component and second component m ay be of the types described above.

[0062] The relative movement between the sample and the substrate m ay be as described above. This relative movement m ay be provided in any number of m anners, such as dipping of the substrate into the sample, the use of pumps or the like for generating the sample movement, relative to the substrate, desired. Clearly, yet another aspect of the invention will relate to an assem bly of the substrate and a moving element causing the relative movement between the sam ple and the substrate. The moving element m ay engage and move the substrate where the sam ple m ay be more or less stationary, or the moving element may move the sam ple where the sam ple is more or less stationary. The moving element may be configured to handle and move the substrate or m ay be pum ps or the like configured to move a fluid sam ple.

[0063] As m entioned, the sample feed line is preferably unblocked. This may be at all points in time (and thus also at manufacture) . All feed lines will present a flow resistance but in this context, an unblocked feed line will allow passage of sample even at the rotation frequency of the first step in the feeding step. Thus, if desired, the assembly m ay be manufactured with a feed line which is blocked, as long as the blocking is overcom e by the sample flow even during the first step of the feeding step.

[0064] BRI EF DESCRI PTI ON OF THE DRAWI NGS

[0065] The invention will now be described in further details with reference to the accom panying drawings, in which:

[0066] Fig. 1 illustrates a first embodim ent of an assem bly for com ponent analysis.

[0067] Fig. 2 illustrates a detailed view of the assem bly from Fig. 1.

[0068] Fig. 3. illustrates a substrate with different wetted areas.

[0069] Fig. 4 illustrates a cross section through the sample cham ber of Fig. 1 with a preferred type of substrate.

[0070] Fig. 5 illustrates a substrate type suitable for SERS measurement.

[0071] Fig. 6 illustrates the substrate of figure 5 in a sample cham ber,

[0072] Fig. 7 illustrates an alternative assembly for the first com ponent analysis.

[0073] Fig. 8 illustrates yet another alternative assem bly for the first component analysis.

[0074] Fig. 9 illustrates a non-rotational m anner of introducing and removing sam ple from the sample chamber. Fig. 10 illustrates a technology not requiring a sam ple cham ber.

[0075] Fig. 1 1 illustrates an analysis of the first component on the substrate.

[0076] Fig. 12 illustrates a second em bodim ent for com ponent analysis.

[0077] Fig. 13 I llustrates tests m ade with human serum and MTX with different types of pretreatm ent of the sam ple before introduction on the substrate.

[0078] Fig. 14 illustrates a com parison between the prior art one-step sam ple introduction and an em bodiment of the step-by-step introduction using MTX as the analyte.

[0079] Fig. 15 illustrates a com parison between the prior art one-step sam ple introduction and an em bodiment of the step-by-step introduction using LTG as the analyte.

[0080] Fig. 16 illustrates the spectral profiles and molecular separation, using MER as the analyte, at different molecular concentrations.

[0081] Fig. 17 illustrates different settlem ent areas for different molecules on the SERS substrate.

[0082] While the invention is susceptible to various modifications and alternative forms, specific embodim ents have been shown by way of example in the drawings and will be described in detail herein. It should be understood, however, that the following description relates to exam ples of embodim ents, and the invention is not intended to be lim ited to the particular forms disclosed. Rather, the invention covers all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims. Furthermore, all the drawings are not to scale, and therefore any ratio extracted from the drawings is not relevant.

[0083] DETAI LED DI SCLOSURE OF THE I NVENTI ON

[0084] I n its most general aspect, the invention relates to a method for analysing a first component of a sam ple com prising the first com ponent and a second component. The m ethod com prises delivering the sam ple to the substrate in steps causing a separation of the second component and the first com ponent over the surface of the substrate. This separation is caused by the forces, and thus acceleration, acting on the first com ponent and the second com ponent during the steps causing the first com ponent to dislodge from any attachm ent to the substrate to a larger degree than the second component. Fig. 1 illustrates an assembly 100 with 10 units 101 for the analysis and a rotating device 102. Each unit 101 com prises 4 chambers: a loading cham ber 103, a sam ple reservoir 104, a sample chamber 105 which com prises a substrate, and a waste chamber 106. An axis z indicates the predeterm ined axis of the rotation of the assem bly. The predeterm ined axis z is passing through a centre of the assembly. The rotating device 102 is configured to rotate the substrate around the predeterm ined axis z, and therefore the unit 101 , with different rotational frequencies. The cham bers are positioned such that the loading cham ber 103 is the closest to the predeterm ined axis z. The loading chamber 103 is followed by the sam ple reservoir 104 placed further towards the outer part of the rotating device. The waste chamber 106 is positioned in the most outer part of the rotating device.

[0085] Fig. 2 illustrates a part of the assem bly 100 shown in Fig. 1. Three entire units 101 are shown in Fig. 2. A substrate 105a is placed in the sample chamber 105. The sam ple cham ber 105 is closer to the centre of the rotating device com pared to the waste cham ber 106. At first, a sample is loaded into a unit 101 through the loading chamber 103. By rotation of the assem bly 100 with a starting rotational frequency around the predeterm ined axis z, i.e. by a starting centrifugal force created, the sample is transported towards the sam ple cham ber 105. Nam ely, from the loading cham ber 103, the sam ple starts to travel through a capillary 107 which connects the loading chamber 103 and the sample reservoir 104. Preferably, the sample reservoir 104 is connected to a vent 108, which is in contact with atmospheric pressure so that no gas resistance is caused by the gas which can be com pressed by the sample when filling-up the sam ple reservoir if there is no vent. Therefore, the sample can freely move and fill-up the sample reservoir 104. The starting rotational frequency which can cause the sam ple transfer depends on the number of param eters, such as the diameter of the capillary 107, the viscosity and density of the sam ple, etc. A typical value of this starting frequency could be 30 Hz. With a sam ple accum ulation in reservoir 104 at a certain point, the starting centrifugal force overcom es the capillary force in the capillary 107, and the sam ple will start to travel towards and through a second conduit, i.e. bottom capillary 109 which extends away from the predeterm ined axis z.

[0086] A part of the sample will reach the sam ple cham ber 105 without touching the substrate 105a. The sam ple cham ber 105 has an input or opening 105o at the end farthest from the predeterm ined axis z. This opening 105o is the only opening of the sample chamber. When the sam ple chamber does not com prise any vents, addition of the sam ple therein would increase the gas pressure therein. The opening of the sam ple chamber is connected to a first conduit 109a. Preferably, the first conduit 109a has an “s” shape having a first and second bend. The first bend is connected to the opening of the sample chamber 105o, and the second bend is connected to the first bend and a waste cham ber 106. The first bend is positioned further away from the predeterm ined axes z than the sample chamber and the second bend is closer to the predeterm ined axis z than the end of the sam ple chamber (which is closer to the predeterm ined axis z) . The second bend of the first conduit 109a is connected to the waste cham ber 106. The second conduit 109 and the first conduit 109a are connected at the first band of the first conduit 109a. The first conduit 109a is preferably also a capillary. An alternative structure would have a first portion of the conduit extending directly from the sample chamber opening to the “second” bend.

[0087] As the sample starts to travel towards the sam ple cham ber 105, gas gets trapped in sam ple cham ber, as no vents are connected to that cham ber 105. This creates a pressure in the sam ple cham ber 105 which further creates a difference in the level of the sample in the first conduit, i.e. capillary 109a as indicated by 11 and I2, and an opportunity for a precise control of the extent of the sam ple in the sample chamber and in the direction toward the axis Z.

[0088] For any higher rotation frequency, the sample will be forced into the sample chamber and thus extend over a portion of the substrate. When the rotation frequency increases, more sam ple will enter the sample cham ber and the farther will the sam ple extend in the direction toward the axis Z. Thus, the rotation frequency determ ines this extent.

[0089] I n Figure 3, a substrate 105a is illustrated. The direction of the arrow is toward the axis Z. A number of dashed lines, L, are provided indicating different portions of the substrate. When rotating the assem bly 100 at a lower rotational frequency, the sam ple may extend to the lowest dashed line. I ncreasing the rotation frequency may m ake the sam ple now extend to the next dashed line and so on.

[0090] I n the present sample, a first component and a second com ponent is present. When the sam ple is forced with a sufficient acceleration over the surface of the substrate 105a, the first com ponent will tend to move with the sam ple to a larger degree than the second component. The second component m ay adhere to the surface of the substrate to a larger degree than the first com ponent. As described above, the reason for this tendency m ay be due to chem ical properties of the com ponents and the substrate and / or m echanical forces acting between them .

[0091] Thus, when the sample moves from one dashed line in Figure 3 to the next, the concentration of the first component in the newly wetted area of the substrate will be relatively larger than in the former wetted portions and the concentration of the first com ponent may be relatively larger than that of the second component. This is illustrated in Figure 4 illustrating the sample extending to two adjacent lines in figure 3, where the extent of the liquid sam ple is illustrated by the waved line L, where the first com ponent is illustrated at 1 and the second com ponent is denoted 2.

[0092] It is seen that as the sample is moved upwardly, in the drawing, a relatively larger num ber of the first com ponents 1 are moved upwardly com pared to the num ber of second com ponents 2. Naturally, when introducing more sample into the sam ple cham ber, more first com ponents 1 and second com ponents 2 are introduced, so that the total number of first components 1 and components 2 will increase. However, by the function of the movement of the sample, and potentially also the affinity of the first com ponent to the surface of the substrate, the concentrations of components 1 at the top of the sam ple extent will be relatively larger.

[0093] It is noted that the tendency of the first com ponent and the second com ponent to move with the sam ple, when moving, will depend on the first component’s and the second component’s adherence, attraction, affinity or the like to the substrate. Naturally, adherence and attraction may be caused by a num ber of factors, such as the size of the components, the chem ical nature or type thereof and a surface and even structure of the substrate.

[0094] Adherence may simply be a stickiness property of the com ponents to the substrate. Protein, fat globules and the like may be sticky in themselves and m ay therefore be prone to im m ediately stick to any surface and may cover the whole surface after im mediate adherence on the surface, creating several layers of the second com ponent around the nanostructured surface and thus present resistance to detach due to the moving sample.

[0095] Adherence may also be influenced by several factors related both to the nature of the substrate and the sam ple. The m etallic type, size and shape and the hydrophobic / hydrophilic properties of the m etallic substrate m ight affect the adherence, while chem ical composition, solvent, pH value, size of the biomolecules and type of aggregation m ight be relevant for the nature of the sample that causes adherence or sickness to the surface.

[0096] A further manner of adhering or attracting may be physical obstruction or blocking of the movem ent of especially the second com ponent during movem ent of the sam ple. An example of this is seen in figure 6, described further below, where the substrate of figure 5 is seen in the sam ple cham ber 105. Figure 6 illustrates a cross section through one of the lines L, so that the sam ple flow is in the direction into and out of the drawing.

[0097] Naturally, any num ber of types of adherence and attraction may be in play sim ultaneously. I n figure 5, the same is illustrated for a preferred type of substrate which is very suitable for a SERS analysis. This substrate 105a com prises a base 301 and a plurality of upright standing pillars 302 with tips 303 with a surface of a SERS active m aterial. The upright standing pillars 302 may be m ade of fused silica while the tips 303 are typically made of a metal, such as gold, silver, alum inium , copper or platinum , or a sem iconductor material, such as one from a group of 111 - V sem iconductors.

[0098] Once the sam ple reaches the desired upper position of the substrate 105a, a relatively large concentration of the first component 1 is seen at the latest wetted portion. This portion maybe called a sensing area 3 and will be used for the analysis.

[0099] I n relation to figure 6, it is seen that the sam ple will flow both over and between the pillars 302, which on the one hand gives the substrate a large surface for the first com ponent and the second com ponent to attach to, and on the other hand forms a grid-like structure which may additionally physically filter the second com ponent if the second com ponent has a size com parable to the distance between the pillars, such as if the second component has a largest dimension which is 10% or more of the inter pillar distance. The inter pillar distance may be the sm allest distance between two neighbouring pillars, a mean distance, a distance from a central axis of the pillars or the like.

[0100] Another interesting factor is the relation between the height, h, of the substrate and the height, H, of the sample container (the inner space thereof in which the substrate is provided) . When h / H is larger than 0.3, such as larger than 0.5, such as larger than 0.75. a relatively large amount of sam ple will flow between the pillars and thus experience this larger surface and physical filtering, whereby a larger proportion of the second component will be retained relatively closer to the sample opening 105o.

[0101] Naturally, the sam e effect m ay be obtained when the substrate has other types of open structures through which the sample m ay flow, such as open porosity, ridges, channels, holes, openings, cavities, or the like. The structure m ay com prise narrow or narrower passages aimed at catching or engaging the second component while allowing the first com ponent to pass.

[0102] Before analysis, it may be desired that no sam ple liquid is present in the sensing area. Thus, it m ay be desired to remove at least a part of the sample rem aining at the sam ple cham ber. This may be achieved by tuning the first rotational frequency to a lower value, such as lower than the starting frequency. Typically, the frequency is decreased to a second rotational frequency which m ay be only 5 Hz. I n that case the gas pressure in the sample cham ber 105 together with the second capillary force created by the first conduit 109a will overcome the first capillary forces and it will push at least a portion of the sam ple rem aining at the sample reception area through the capillary 109a and towards the reservoir 104 and the waste chamber 106 and thus away from the sensing area.

[0103] As an alternative, a vent or second opening m ay be provided in the sam ple cham ber, so that no pressure is built-up in the sample chamber. I n that respect, the evacuation of the sam ple chamber or a part thereof may be taken care of by spinning the disc faster, forcing the liquid outwardly, or by providing other flow channels allowing the liquid to leave the sam ple chamber.

[0104] Fig. 7 illustrates an alternative assembly 100 with 10 units 101 for the analysis and a rotating device 102. Each unit 101 com prises 4 cham bers: a loading cham ber 103, a sample reservoir 104, a sam ple cham ber 105 which comprises a substrate 105a and a waste cham ber 106. This alternative embodiment m ay enable removing the at least a portion of the sample by creating a constant flow of the sample further away from the substrate with respect to the predeterm ined axis. This m ay be achieved with a capillary 109 directly connected from the loading chamber 103 to the waste chamber 106.

[0105] While the assembly 100 is in a stationary position, the sam ple may be injected through the loading chamber 103. The rotation of the assem bly 100 around a predeterm ined axis z m ay be established with the first rotational frequency. The sample may start moving from the loading chamber 103, due to the centrifugal force created, to the sample reservoir 104 and fills the capillary 109. A flow rate of the sam ple depends on the capillary size. The flow rate of the liquid part in the capillary 109 m ay be different from a flow rate in the capillary 109a. If the flow rate in the capillary 109 is larger than the flow rate in the capillary 109a, a level of the liquid part m ay start to rise towards the sam ple cham ber 105 until it reaches equilibrium condition. I n this m anner the level of the liquid part m ay be controlled by varying the first rotational frequency. The first rotational frequency may be tuned so that the sam ple im merses the substrate partially. For instance, the first rotational frequency may be decreased so that the level of the liquid part on the substrate, positioned in the sam ple chamber, is held constant. Optionally, there m ay be a vent 105b in the sample cham ber. Furthermore, as there is the sam ple flow towards the waste chamber 106 through the capillary 109a, the sam ple m ay gradually be transferred to the waste cham ber 106 during the rotation.

[0106] Fig. 8 illustrates yet another alternative assem bly 100 with 10 units 101 for the substrate preparation and a rotating device 102. Each unit 101 com prises 2 chambers: a loading chamber 103 and a sam ple cham ber 105 which com prises a substrate 105a. At first, a sample is loaded into a unit 101 through the loading chamber 103 while the assem bly is in stationary position. A vent 108 is im plemented for ventilation during the sample loading. The rotation of the assembly around the predeterm ined axis z is established with the first rotational frequency. I n this stage the sam ple does not reach the substrate 105a. The sam ple will reach the substrate 105a by tuning the rotational frequency so that it com pensates pressure in the sample chamber 105. One or more tim es, the rotational frequency will be changed to a second rotational frequency typically larger than the first rotational frequency. I n each case the second rotational frequency m ay be tuned so that, in an equilibrium condition, a level of the liquid part of the sam ple in the sam ple cham ber 103 is above the substrate 105a, partially im m ersing the substrate 105a. Finally, the assembly is brought back to stationary position. As there are no more centrifugal forces acting on liquid part of the sample, pressure in the sam ple cham ber 105 pushes the liquid part back to the loading cham ber 103. I n this case the loading chamber 103 has a role of a waste chamber.

[0107] I n figure 9, a m anner of obtaining the same type of sample movement over the substrate 105a is obtained using a pump 6 for pum ping sample into the sample chamber 105 in the same manners as above: stepwise with a predeterm ined acceleration of the sample so that the first com ponents 1 will have a larger tendency than the second com ponents 2 to move with the sample. Finally, a pum p 5 m ay be used for removing at least part of the sam ple at least from the sensing area 3 to prepare the substate for the analysis.

[0108] I n figure 10, the sam e is achieved by dipping the substrate 105a in the sample provided in an open container. The dipping may again be perform ed so that the relative movement between the substrate 105a and the sam ple is as described above. The arrows to the right indicate 3 downward movements to increasing depths in the sam ple and a final removal of the substrate from the sample.

[0109] I n figure 1 1 , analysis of the first com ponents in the sensing area is exem plified by e.g. an optical analysis. Clearly, any type of analysis may be performed. Especially when the substrate type is as seen on figure 3 m ay SERS or Raman-based analysis be preferred.

[0110] Fig. 12 illustrates an alternative assem bly 100’ which has overall the sam e functionality as that of figure 1 . The sam e components with at least substantially the same functionalities have been added a mark (“ ‘ “) compared to figure 1 . Thus, the assem bly 100’ has with 8 units 101 ’ for the analysis and a rotating device 102’. Each unit 101 ’ com prises 4 cham bers: a loading chamber 103’, an additional chamber 103”, a m ixing / sample reservoir 104’, and a sample chamber 105’ which comprises a substrate. An axis z indicates the predeterm ined axis of the rotation of the assem bly. The predeterm ined axis z is passing through a centre of the assem bly. The rotating device 102’ is configured to rotate the substrate around the predeterm ined axis z, and therefore the unit 101 ’, with different rotational frequencies. The chambers are positioned such that the loading chamber 103’ and the additional cham ber 103” are the closest to the predeterm ined axis z. The loading cham ber 103’ and additional chamber 103” are followed by the m ixing / sam ple reservoir 104’ placed further towards the outer part of the rotating device. The sam ple cham ber 105’ is positioned in the most outer part of the rotating device. A substrate 105a is placed in the sam ple cham ber 105’.

[0111] At first, a sam ple is loaded into a unit 101 ’ through the loading cham ber 103’. Another liquid, such as comprising further elements form ing the final sample with the sam ple in cham ber 103’, is added to the additional chamber 103”. The other liquid may com prise the first com ponent if not present in the sam ple provided to chamber 103”. Also other components, such as components altering the hydrophobicity / hydrophilicity of the substrate surface, may be added.

[0112] By rotation of the assem bly 100 with a starting rotational frequency around the predeterm ined axis z, i.e. by a starting centrifugal force created, the sample and other liquid are transported towards the m ixing / sample chamber 105’. Nam ely, from the loading cham ber 103’, the sample starts to travel through a capillary 107 which connects the loading chamber 103’ and the sample reservoir 104’. The sam e is the situation for the other sample

[0113] Preferably, the sample reservoir 104’ is connected to a vent 108’, which is in contact with atmospheric pressure so that no gas resistance is caused by the gas which can be com pressed by the sample when filling-up the sample reservoir if there is no vent. Therefore, the sam ple and other liquid can freely move and fill-up the sam ple reservoir 104’. The starting rotational frequency which can cause the sample transfer depends on the num ber of param eters, such as the diam eter of the capillary 107’, the viscosity and density of the sample, etc. A typical value of this starting frequency could be 30 Hz. When the sam ple and other liquid enters the reservoir 104’, a m ixing m ay be performed. This m ixing m ay comprise a spinning up / down of the disc to facilitate m ixing.

[0114] With a sam ple accum ulation in reservoir 104’ at a certain point, the starting centrifugal force overcom es the capillary force in the capillary 107’, and the sample will start to travel towards and through a second conduit, i.e. bottom capillary 109’ which extends away from the predeterm ined axis z. Then, the above steps are perform ed to introduce the sam ple on to the substrate.

[0115] No separate waste com partm ent is provided, so that spinning-down of the disc will force the sample back into the cham ber 104’.

[0116] Examples I n general, a centrifugal m icrofluidic device, with an outer diam eter of 120 m m and an inner one of 15.2 m m , consists of 8 units and generally as seen in figure 12. Each unit contains a loading chamber for m ethanol and one for serum or pre-treated serum / blood, a m ixing chamber, a pneum atic cham ber where a SERS chip (4 x 4 m m) is glued using PSA, and m icrochannels that connect all reservoirs. All m icrochannels have a width of 0.40 m m . Rotation of the device was achieved with a spin stand using a spindle motor ( RE 35, Maxon motor AG, Sachseln, Switzerland) .

[0117] The SERS substrate was a 4x4m m substrate com prising silica nanopillars with a silver coating where the nanopillars have a height of 400 nm , a width of ~50 nm in width, with a density of ~20 NP / pm2. The substrate is prepared by silicon etching and subsequent coating as is widely known.

[0118] A com mercial serum (from blood) sam ple was spiked with 25 pM of methotrexate (anticancer drug) as a model analyte. A 13 pL of the spiked sam ple was loaded in the loading chamber and m ixed with 65 pL of methanol by providing a rpm of 10 to the device with an acceleration of 10Hz / s.

[0119] Once added to the cham ber 104’, the liquids are m ixed by conveying a 75 Hz / s acceleration to the rotatable element while varying the rpm between 10 and 30 for 1 m inute.

[0120] After the m ixing, the solution was pushed, by increasing the rotation frequency to 30 with an acceleration of 10Hz / s, to the sam ple chamber and a substrate therein.

[0121] The below different m ethods were carried out during the pushing phase and a SERS measurement was conducted by DXRxi Raman m icroscope on the silver nanopillars, where a wavelength of 686 cm - 1 was used for investigating the presence of the drug (Methotrexate) . The below results are in arbitrary units illustrating the amount of the drug at the last wetted portions of the substrate.

[0122] After the steps, the device was rotated to 10Hz with a deceleration of 10Hz / s. Finally, the device was brought to a full stop.

[0123] Example 1 :

[0124] 8 steps were performed with a rotation frequency increase of 5Hz, where the first step starts at 30Hz and the last step ends at 70Hz, with an acceleration of 10Hz / s in each step. Between adjacent steps, a delay of 5 seconds was provided. The recorded intensity was 38 a.u. Example 2:

[0125] A single step was performed with a rotation frequency increase from 10Hz to 55Hz with an acceleration of 10Hz / s. The recorded intensity was 5 a.u.

[0126] Example 3:

[0127] 8 steps were performed with a rotation frequency increase of 5Hz with an acceleration of 1 Hz / s. Between adjacent steps, a delay of 5 seconds was provided. The recorded intensity was 20 a.u.

[0128] Example 4:

[0129] A single step was performed with a rotation frequency increase from 10Hz to 55Hz with an acceleration of 1 Hz / s. The recorded intensity was 19 a.u.

[0130] Figure 13 relates to tests made on a sam ple com prising hum an serum and MTX. Results a) , b) and c) illustrate the steps and the waiting tim e between steps. Result a) relates to a situation where the serum (as a high com plex biological fluid) was first exposed to protein precipitation in the centrifugal m icrofluidics cartridge before being fed to the SERS substrate . Result b) relates to a situation where the sam ple was first exposed to ultrafiltration outside the in the centrifugal m icrofluidics cartridge before being fed to the SERS substate. Result c) relates to a situation where the sample was first exposed to solid-phase extraction outside the in the centrifugal m icrofluidics cartridge before being fed to the SERS substate. Curves d) show the com parative SERS spectral variations of MTX for the band at 686 cm - 1 after each separation m ethod on the disc. Dotted lines represents the spectra of the blank solution (the matrix without the analyte) . It is clear that a useful band of analyte scattering is seen in all three situations.

[0131] Figure 14 illustrates a com parison of the molecular separation of MTX on the SERS chip after perform ing the prior art one-step sample introduction and an em bodiment of the step-by- step centrifugal m icrofluidics separation. Result a) illustrates the intensity localization of MTX on the chip surface after perform ing the one step separation on the disc with a rotation frequency increase from 10Hz to 55Hz with an acceleration of 10Hz / s. Result b) illustrates the intensity localization of MTX on the chip surface after perform ing a step-by-step acceleration and sam ple introduction on the SERS substrate. Result c) shows the com parative SERS spectral variations of MTX for the band at 686 cm - 1 after each method; an improvement in intensity is obtained with the step-by-step introduction of the sam ple on the disc. Figure 15 illustrates a com parison of the molecular separation of LTG on the SERS chip, which in this exam ple has a gold surface, after perform ing the prior art one-step sam ple introduction and an em bodim ent of the step-by-step centrifugal m icrofluidics separation. Result a) illustrates the intensity localization of LTG on the chip surface after perform ing one step separation on the disc; a rotation frequency increase from 10Hz to 55Hz with an acceleration of 10Hz / s. Result b) illustrates the intensity localization of LTG on the chip surface after perform ing a step-by-step separation on disc. Result c) shows the comparative SERS spectral variations of LTG for the band at 1356 cm- 1 after each method; an im provem ent in intensity and less background contribution is obtained with the step-by-step introduction of the sam ple on the disc.

[0132] Figure 16 illustrates a com parison of the MER (Meropenem) spectral profile and SERS scanning on the SERS chip after perform ing the disc separation with the indicated frequency steps and waiting tim es. Result a) is a comparison of intensity profile and analyte localization of MER at different concentrations spiked in serum and the separated and “filtrated” on the SERS chip. Result b) is a com parison of the spectral profile of MER at the different concentrations. It is seen that the band at 1559 cm - 1 becomes more intense when increasing the concentration.

[0133] Figure 17 illustrates another improvement obtained by the step-by-step method in relation to the separation of sm all molecules: result a) shows to the left the separation of uric acid (sm all molecule) at the bottom of the chip; it is seen that there is an adsorption im m ediately after reaching the substrate. Result a) to the right illustrates MTX (a molecule larger than uric acid) being separated and retained at the upper and m iddle part of the sam e chip. Result b) illustrates the average spectral profile of the upper (unbroken line) and lower (dotted line) regions in the chip. A high band intensity is for uric acid in the low region, while a high band intensity is for MTX in the upper-m iddle region

Claims

CLAI MS1 . A method of analyzing a first com ponent of a fluid sample com prising the first component and a second component, the method com prising: providing a rotatable elem ent having an axis of rotation com prising: o a sam ple cham ber com prising a sample inlet positioned farther away from the axis of rotation than a portion of the sam ple cham ber, o a substrate provided at least partly in the portion of the sample chamber, o a sam ple receptacle positioned closer to the axis of rotation than the sam ple inlet and o a sam ple feed line from the sample receptacle to the sample inlet providing the sam ple in the sam ple receptacle, feeding sample into the sam ple cham ber by, in each step of a sequence of steps, increasing a rotation frequency of the rotatable element, removing sam ple from at least a first portion of the substrate having received sam ple during the last step of the sequence of steps, and perform ing a quantitative analysis of the first component in the first portion, where: each step of the sequence of steps comprises increasing the rotation frequency from a first rotation frequency to a second rotation frequency within no more than 1 s and with a rotation frequency increase of at least 1 Hz.

2. A m ethod according to claim 1 , wherein a delay of 1 - 100s is provided between two adjacent steps.

3. A m ethod according to claim 1 or 2, wherein, during at least a portion of a step, the sam ple moves, relative to the substrate, with an acceleration of 0.1 -10mm / s2.

4. A m ethod according to any of the preceding claims, wherein: the feeding step com prises, in each step of the sequence of steps, increasing a gas pressure inside the sam ple cham ber and the removing step com prises the increased gas pressure forcing the at least portion of the sample out of the sam ple cham ber.

5. A m ethod according to claim 6, wherein the removing step com prises reducing a rotation frequency of the rotatable element.

6. A m ethod according to any of the preceding claims, wherein the removing step comprises providing a suction force acting from the sam ple chamber and towards a waste cham ber.

7. A m ethod according to any of the preceding claims, wherein the feeding step com prises feeding sam ple into the sample container in each feeding step.

8. A m ethod according to any of the preceding claims, wherein the sample feed line allows sam ple flow during all feeding steps.

9. An assembly com prising a rotating device, an analyzer, and a rotatable element having an axis of rotation, wherein the rotatable element com prises: a sample chamber comprising a sample inlet positioned farther away from the axis of rotation than a portion of the sample chamber, a substrate provided at least partly in the portion of the sam ple chamber, a sample receptacle positioned closer to the axis of rotation than the sam ple inlet and a sample feed line from the sample receptacle to the sample inlet and wherein the rotating device is configured to rotate the rotatable elem ent around the predeterm ined axis to:feed sam ple from the sample inlet to the sample chamber by, in each step of a sequence of steps, increasing a rotation frequency of the rotatable elem ent, each step of the sequence of steps com prising increasing the rotation frequency from a first rotation frequency to a second rotation frequency within no more than 1 s and with a rotation frequency increase of at least 1 Hz and remove sam ple from at least a first portion of the substrate having received sam ple during the last step of the sequence of steps, and wherein the analyzer is configured to perform a quantitative analysis of the first component in the first portion.

10. An assembly according to claim 9, wherein a delay of 1 - 100s is provided between two adjacent steps.1 1 . An assembly according to claim 9 or 10, wherein, during at least a portion of a step, the sample moves, relative to the substrate, with an acceleration of 0.1 - 10m m / s2.

12. An assembly according to any of claims 9- 1 1 , wherein the substrate com prises a base with pillars extending therefrom , the pillars being spatially displaced from each other, the pillars having a first end attached to the base and a second end.

13. An assembly according to claim 12, wherein the sample chamber has an internal height of no more than 10 tim es a height of the pillars.

14. An assembly according to any of claims 9- 13, wherein the sample feed line is unblocked.

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