WO2003034041A1

WO2003034041A1 - Particle measurement system using time-frequency transform

Info

Publication number: WO2003034041A1
Application number: PCT/GB2001/004552
Authority: WO
Inventors: Andreas Manz; Yien Chian Kwok
Original assignee: Imperial College Innovations Ltd
Current assignee: Ip2ipo Innovations Ltd
Priority date: 2001-10-12
Filing date: 2001-10-12
Publication date: 2003-04-24
Anticipated expiration: 2004-04-12

Abstract

A measurement system for and method of characterizing particles of a fluid sample, the system comprising: a microfluidic chip including a migration channel through which particles of a fluid sample are driven in a fluid medium; an illumination unit for illuminating at least a plurality of spaced sections in a detection region of the migration channel; an optical detector for detecting optical emission from at least the plurality of spaced sections in the detection region of the migration channel; an acquisition unit for generating a time-domain signal from the optical emission as detected; and a processing unit operably coupled to the acquisition unit for receiving the time-domain signal and transforming the time-domain signal to a frequency-domain signal, which frequency-domain signal provides for characterization of the particles.

Description

PARTICLE MEASUREMENT SYSTEM USING TIME-FREQUENCY TRANSFORM

The present invention relates to a measurement system for and method of characterizing particles of a fluid sample, in particular measuring velocities of particles in a fluid medium and numbers of particles.

The characterization of particles of a fluid sample, for example, from a determination of the velocities of particles in a liquid or gaseous medium, is of increasing importance, in particular in biology and industrial processes. In biology, numerous solid-liquid systems exist, such as blood, which require characterization. Also, in biology, the measurement of the velocities of cells moving under the influence of an electric field is important. The intrinsic electrophoretic mobilities (EPMs) of cells could reveal whether cells under different physiological conditions or exposed to different physiologically- active agents would have an altered net surface charge as reflected by their different EPMs.

Recently, a measurement technique has been developed, which utilizes the convolution of a detection function, as a Shah function, followed by a Fourier transform, for measuring the velocities of solute plugs injected into the separation channel of a microfluidic chip (H John Crabtree, Martin U Kopp and Andreas Manz, Anal. Chem. 1999, 71, pages 2130 to 2138).

The present inventors have now recognized that this measurement technique can be adapted and extended to characterize particles of fluid samples without requiring the injection of discrete plugs of fluid samples.

It is thus an aim of the present invention to provide a measurement system for and method of characterizing particles of fluid samples.

Accordingly, the present invention provides a measurement system for characterizing particles of a fluid sample, comprising: a microfluidic chip including a migration channel through which particles of a fluid sample are driven in a fluid medium; an illumination unit for illuminating at least a plurality of spaced sections in a detection region of the migration channel; an optical detector for detecting optical emission from at least the plurality of spaced sections in the detection region of the migration channel; an acquisition unit for generating a time-domain signal from the optical emission as detected; and a processing unit operably coupled to the acquisition unit for receiving the time-domain signal and transforming the time-domain signal to a frequency-domain signal, which frequency-domain signal provides for characterization of the particles.

Preferably, the system provides for measurement of velocities of particles in a fluid medium.

Preferably, the system provides for measurement of numbers of particles.

In one embodiment the particles are transported in a fluid flow through the migration channel.

In another embodiment the particles are driven through a fluid medium in the migration channel.

In one preferred embodiment the particles are pressure driven through the migration channel.

In another preferred embodiment the particles are driven through the migration channel by hydrodynamic chromatography.

In a further preferred embodiment the particles are driven through the migration channel by flow-field fractionation.

In yet another preferred embodiment the particles are driven through the migration channel by electrophoresis.

Preferably, the electrophoresis is dielectrophoresis. In still yet another preferred embodiment the particles are driven through the migration channel by hydrodynamic pumping.

In one embodiment the hydrodynamic pumping is electrohydrodynamic pumping.

In another embodiment the hydrodynamic pumping is magnetohydrodynamic pumping.

In one embodiment the optical detector is a fluorescence detector for detecting fluorescence of the particles.

In another embodiment the optical detector is a light-scattering detector for detecting light scattering from the particles.

In a further embodiment the optical detector is an absorption detector for detecting optical absorption by the particles .

In one embodiment the illumination unit includes a slit array comprising a plurality of spaced windows disposed in registration with the plurality of spaced sections in the detection region of the migration channel, and a light source for providing a light beam directed onto the slit array.

Preferably, the slit array is integrated with the microfluidic chip.

Preferably, the light source is a laser.

In another embodiment the illumination unit comprises a plurality of spaced light elements for illuminating the plurality of spaced sections in the detection region of the migration channel.

Preferably, the illumination unit comprises at least one light source and a plurality of light-transmitting elements coupled to the at least one light source and disposed in spaced relation to the detection region in the migration channel such as to illuminate respective ones of the plurality of spaced sections in the detection region of the migration channel.

More preferably, the light-transmitting elements are integrated with the microfluidic chip.

In one embodiment the light-transmitting elements comprise holographic lenses.

In another embodiment the light-transmitting elements comprise waveguides.

In a further embodiment the light-transmitting elements comprise optical fibers.

Preferably, the at least one light source is a laser.

Preferably, the illumination unit comprises a plurality of light sources, each coupled to a respective one of the light-transmitting elements.

In one embodiment the optical detector comprises a single detector for detecting optical emission from each of the plurality of spaced sections in the detection region of the migration channel.

In another embodiment the optical detector comprises a plurality of spaced detector elements for detecting optical emission from respective ones of the plurality of spaced sections in the detection region of the migration channel.

Preferably, the detector elements comprise photodetectors.

In a further embodiment the illumination unit comprises a single light source for illuminating the detection region of the migration channel, and the optical detector comprises a plurality of spaced detector elements for detecting optical emission from a plurality of spaced sections in the detection region of the migration channel.

Preferably, the light source is a laser. Preferably, the detector elements comprise photodetectors.

Preferably, the system further comprises: a dilution stage for performing dilution of the fluid sample such that the concentration of particles is less than a predetermined concentration.

In one embodiment the dilution stage is configured to provide that the concentration of particles is such that the average spacing of particles driven through the migration channel is not less than the center-to-center spacing of adjacent ones of the plurality of spaced sections in the detection region of the migration channel.

In another embodiment the dilution stage is configured to provide that the concentration of particles is such that the average spacing of particles driven through the migration channel is not less than about 0.5 mm.

Preferably, the dilution stage is configured to provide that the concentration of particles is such that the average spacing of particles driven through the migration channel is not less than about 1 mm.

Preferably, the dilution stage is operably controlled by the processing unit such as to provide for the necessary dilution of the fluid sample.

In one embodiment the transform is a Fourier transform.

In another embodiment the transform is a wavelet transform.

Preferably, the transform is a continuous wavelet transform.

In one embodiment the particles comprise single molecules.

In another embodiment the particles comprise particles of a particle-based assay. Preferably, the assay is an immunoassay.

In a further embodiment the particles comprise biological cells.

In one preferred embodiment the particles comprise blood cells, namely red or white blood cells.

In another preferred embodiment the particles comprise viruses.

In a further preferred embodiment the particles comprise bacteria.

The present invention also provides a method of characterizing particles of a fluid sample, comprising the steps of: driving particles of a fluid sample through a migration channel of a microfluidic chip; illuminating at least a plurality of spaced sections in a detection region of the migration channel; detecting optical emission from at least the plurality of spaced sections in the detection region of the migration channel; generating a time-domain signal from the optical emission as detected; and transforming the time- domain signal to a frequency-domain signal, which frequency-domain signal provides for characterization of the particles.

Preferably, the characterization provides for measurement of velocities of particles in a fluid medium.

Preferably, the characterization provides for measurement of numbers of particles.

In one preferred embodiment the particles are pressure driven through the migration channel. In another preferred embodiment the particles are driven through the migration channel by hydrodynamic chromatography.

Preferably, the electrophoresis is dielectrophoresis.

In still yet another preferred embodiment the particles are driven through the migration channel by hydrodynamic pumping.

In one embodiment the hydrodynamic pumping is electrohydrodynamic pumping.

In another embodiment the hydrodynamic pumping is magnetohydrodynamic pumping.

In one embodiment the step of detecting optical emission comprises the step of detecting fluorescence of the particles.

In another embodiment the step of detecting optical emission comprises the step of detecting light scattering from the particles.

In a further embodiment the step of detecting optical emission comprises the step of detecting optical absorption by the particles.

In one embodiment the step of illuminating at least a plurality of spaced sections in the detection region of the migration channel comprises the step of illuminating a plurality of spaced sections in the detection region of the migration channel.

Preferably, illumination is from a laser. In one embodiment the step of detecting optical emission comprises the step of detecting optical emission from each of the plurality of spaced sections in the detection region of the migration channel using a single detector.

In another embodiment the step of detecting optical emission comprises the step of detecting optical emission from the plurality of spaced sections in the detection region of the migration channel using a plurality of spaced detector elements, each detecting optical emission from a respective one of the plurality of spaced sections in the detection region of the migration channel.

In a further embodiment the step of illuminating at least a plurality of spaced sections in the detection region of the migration channel comprises the step of illuminating the detection region of the migration channel, and the step of detecting optical emission comprises the step of detecting optical emission from a plurality of spaced sections in the detection region of the migration channel using a plurality of spaced detector elements, each detecting optical emission from a respective one of the plurality of spaced sections in the detection region of the migration channel.

Preferably, illumination is from a laser.

Preferably, the method further comprises the step of: diluting the concentration of particles in the fluid sample such that the concentration of particles is less than a predetermined concentration.

In one embodiment the concentration of particles is such that the average spacing of particles driven through the migration channel is not less than the center-to-center spacing of adj acent ones of the plurality of spaced sections in the detection region of the migration channel.

In another embodiment the concentration of particles is such that the average spacing of particles driven through the migration channel is not less than about 0.5 mm. Preferably, the concentration of particles is such that the average spacing of particles driven through the migration channel is not less than about 1 mm.

Preferably, the dilution of the fluid sample is in response to the generated time-domain signal.

In one embodiment the transform is a Fourier transform.

In another embodiment the transform is a wavelet transform.

Preferably, the transform is a continuous wavelet transform.

In one embodiment the particles comprise single molecules.

In another embodiment the particles comprise particles of a particle-based assay.

Preferably, the assay is an immunoassay.

In a further embodiment the particles comprise biological cells.

In one preferred embodiment the particles comprise blood cells, namely red and white blood cells. ^•

In another preferred embodiment the particles comprise viruses.

In a further preferred embodiment the particles comprise bacteria.

In one embodiment the fluid sample comprises a single fluid sample including a plurality of particles of different kind, whereby the method provides for characterization of each different kind of particle.

In a preferred embodiment the spaced sections in the detection region of the migration channel are equi-spaced. In one embodiment the wavelength of the illuminating radiation is in the visible spectrum.

In another embodiment the wavelength of the illuminating radiation is outside the visible spectrum.

Preferred embodiments of the present invention will now be described hereinbelow by way of example only with reference to the accompanying drawings, in which:

Figure 1 schematically illustrates a measurement system in accordance with a preferred embodiment of the present invention;

Figure 2 illustrates the relative positions of the light source and the optical detector of the measurement system of Figure 1 ;

Figure 3 illustrates a plan view of the layout of the chip of the microfabricated particle migration unit of the measurement system of Figure 1;

Figure 4 illustrates in enlarged scale the slit array of the chip of Figure 3;

Figure 5 illustrates part of the time-domain signal as acquired by the measurement system of Figure 1 in the described Example;

Figure 6(a) illustrates the frequency-domain magnitude plot of the Fourier transform of the time-domain signal as acquired by the measurement system of Figure 1 in the described Example;

Figure 6(b) illustrates in enlarged scale the frequency-domain magnitude plot encompassing the fundamental peak at 7.1 Hz;

Figure 7(a) illustrates the time-domain signal as acquired by the measurement system of Figure 1 in the described Example; Figure 7(b) illustrates a plot of the wavelet transform in the frequency region about 7 Hz of the time-domain signal as acquired by the measurement system of Figure 1 in the described Example; and

Figure 8 illustrates part of a modified optical detector for the measurement system of Figure 1.

The measurement system comprises a microfabricated particle migration unit 1, in this embodiment fabricated as a substrate chip, through which particles of a fluid sample, in this embodiment a liquid sample, are driven.

The particle migration unit 1, in this embodiment an electrophoresis chip, includes a migration channel 5, in this embodiment an elongate linear channel having a length of 57 mm, through which particles of a fluid sample are driven, a sample reservoir 7 for receiving a volume of a fluid sample, a sample reservoir channel 8, in this embodiment having a length of 5 mm, fluidly connecting the sample reservoir 7 and one end of the migration channel 5, a buffer reservoir 9 for receiving a volume of a buffer solution, a buffer reservoir channel 10, in this embodiment having a length of 37 mm, fluidly connecting the buffer reservoir 9 and the one end of the migration channel 5, a sample waste reservoir 11 for receiving a volume of waste fluid sample, a sample waste channel 12, in this embodiment having a length of 43 mm, fluidly connecting the sample waste reservoir 11 and the one end of the migration channel 5, and a buffer waste reservoir 13 for receiving a volume of waste buffer solution fluidly connected to the other end of the migration channel 5.

The particle migration unit 1 is fabricated from three planar substrate plates, in this embodiment a first, lower plate composed of microsheet glass, a second, intermediate plate composed of poly (dimethylsiloxane) (PMDS), and a third, upper plate composed of microsheet glass. In a first step, the first plate was etched to form wells which define the migration channel 5, the sample reservoir channel 8, the buffer reservoir channel 10 and the sample waste channel 12. In this embodiment the wells have a depth of 10.5 μm, a width of 15 μm at the bottom thereof and a width of 36 μm at the top thereof. In a second step, four holes were bored into the second sheet, in this embodiment having a thickness of 2 mm, so as to provide openings having a diameter of 2 mm which define the sample reservoir 1, the buffer reservoir 9, the sample waste reservoir 11 and the buffer waste reservoir 13. In a third step, a slit array 15 was fabricated on the third plate, in this embodiment having a thickness of 2.5 mm. In this embodiment the slit array 15 was fabricated by depositing a chromium film having a thickness of 100 nm onto the third plate and patterning the chromium film to provide 375 equi-spaced detection windows 17, each having a width w of 40 μm and a center-to-center spacing d of 70 μm. In a fourth and final step, the plates were assembled such that the openings in the second plate were aligned with the wells in the first plate and the slit array 15 on the third plate was aligned with substantially the mid point of the migration channel 5.

The measurement system further comprises a light source 19 for providing a light beam, and a lens arrangement 21 for expanding the light beam, in this embodiment as a linear beam, and directing the expanded light beam onto a region of the migration channel 5 in the particle migration unit 1 ; this region of the migration channel 5 being the detection region.

In this embodiment the light source 19 comprises an argon ion laser (line at 488 nm; model 532-B-A01, OmNichrome; Melles Griot, Chino, California, US).

In this embodiment the lens arrangement 21 comprises at least one concave cylindrical lens 23, in this embodiment a single lens (a Powell- 10-0.75 lens (Elliot Scientific, Hertfordshire, UK)), for expanding the light beam, and at least one convex cylindrical lens 25 (01LCP009-/= 80 mm; Melles Griot), spaced from the concave lens 23 and the particle migration unit 1, in this embodiment by 4.5 cm and 7.5 cm respectively, for providing a parallel beam expanded along one dimension to 2 cm and having a width of 100 μm.

In this embodiment the light source 19, the lenses 23, 25 of the lens arrangement 21 and the particle migration unit 1 are supported on a vertically-mounted optical rail (not illustrated) (lens mounts and posts on a 2 m X-95 rail and carrier system; Newport, Irvine, California, US). The measurement system further comprises an optical detector 27 for detecting the migration of particles through the migration channel 5 of the particle migration unit 1, in this embodiment by detecting the optical emission of the particles. In this embodiment, as illustrated in Figure 2, the optical detector 27 is disposed at an angle from the plane of the expanded light beam, here 30 degrees, and spaced from the detection region of the particle migration unit 1, here by 3.5 cm.

In this embodiment the optical detector 27 comprises a photomultiplier tube (PMT), specifically a 5.1 cm diameter head-on PMT (R550 PMT, El 198-11 socket, C3830 power supply; Hamamatsu Photonics, Middlesex, UK), and associated filters, specifically at least one high-pass interference filter, in this embodiment three high-pass interference filters (505EFLP; Omega Optical, Brattleboro, NT, US), at least one Schott filter, in this embodiment three high-pass Schott filters (OG515; Edmund Scientific, Barrington, New Jersey, US), and at least one emission band-pass filter, in this embodiment one fluoroscein emission band-pass filter (520DF15; Omega Optical); the latter filter being disposed downstream of the other filters. The edges of the filters and the PMT are foil wrapped to prevent unfiltered light from reaching the PMT.

The measurement system further comprises a data acquisition unit 29 which is connected to the optical detector 27 for logging the output signal thereof. In this embodiment the data acquisition unit 29 comprises a PICO analog-to-digital converter data acquisition unit (ADC; Pico Instruments) having a scan rate set at 100 Hz, and the current signal output from the PMT biased at -1000 V is filtered with a low-pass filter (NBF21 M; Kemo, Kent, UK) set at a 40 Hz cut-off frequency.

The measurement system further comprises a power supply 31 for applying potentials at the electrodes in each of the sample reservoir 7, the buffer reservoir 9, the sample waste reservoir 11 and the buffer waste reservoir 13. In this embodiment the power supply 31 comprises four multiplexed discrete dc-dc converters.

The measurement system further comprises a sample dilution stage 33 for diluting a fluid sample to be measured as necessary; no dilution being necessary where the concentration of particles in the fluid sample is sufficiently low. By ensuring that the fluid sample is sufficiently dilute, the particles in the fluid sample should be driven independently through the migration channel 5 of the particle migration unit 1, thereby enabling the detection of the migration of each separate particle. In a preferred embodiment the concentration of particles in a fluid sample should be such that the average spacing of the particles migrating through the migration channel 5 is about 1 mm.

The measurement system further comprises a processing unit 35, in this embodiment a personal computer, for controlling the power supply 31 and the dilution stage 33, in this embodiment from a LabNiew program (National Instruments, Austin, Texas, US), and operating on the acquired data. In one embodiment the dilution stage 33 is controlled by a continuous feedback loop such as to increasingly dilute the fluid sample where the acquired time-domain signal does not have the required resolution. Fast Fourier transforms (FFTs) are performed using Igor Pro 3 (Wavemetrics, Lake Oswego, Oregon, US) with Microsoft Excel 5.0 after acquisition. Wavelet transforms (WTs) are performed using the wavelet toolbox of Matlab (The Mathworks Inc., Natick, MA, US).

The components of the measurement system are disposed in a light-tight box, in this embodiment a galvanized steel box.

Operation of the measurement system of the present invention will now be described with reference to the following non-limiting Example.

Reagents and Solutions

A Tris-Borate-EDTA (TBE) buffer solution was prepared at O.lx concentration (8.9 mM each of tris-(methoxy)aminomethane and boric acid, 0.2 mM in ethylenediaminetetraacetic acid; prepared from a solid TBE mixture (Fluka, Buchs, Switzerland)) with de-ionized water and filtered through 0.2 mm filters (Millisart^®). The use of a TBE buffer solution has been found to almost completely eliminate both particle aggregation and adhesion of the particles to the channel walls. A 150 μM fluoroscein in O.lx TBE solution was prepared by dissolving an appropriate amount of sodium fluoroscein (salt, Fluka) in the O.lx TBE solution.

Fluorescent polystyrene microspheres, amine modified, 1 μm diameter, yellow-green (505/515), 2 % solids (Molecular Probes Europe BN, The Netherlands).

A cleaning solution of 0.5 M sodium hydroxide was prepared from de-ionized water and sodium hydroxide (BDH, Poole, UK).

A 0.002 % (-3.6 x 10⁶ microspheres/mL) fluorescent microspheres sample solution was prepared by diluting the stock fluorescent polystyrene microspheres sample (2 %) by 1000-fold with the O.lx TBE solution.

Experimental

In this Example, only 50 of the 375 windows 17 in the slit array 15 on the particle migration unit 1 were used. The remaimng windows 17 were blocked, in this embodiment with aluminium foil.

The particle migration unit 1 was prepared by first drawing an amount of the cleaning solution and then the TBE buffer solution thereinto. In this Example, the cleaning solution and the TBE buffer solution were drawn into the particle migration unit 1 by applying a vacuum to one of the reservoirs 7, 9, 11, 13 and supplying first the cleaning solution and then the TBE buffer solution to the other of the reservoirs 7, 9, 11, 13.

The sample reservoir 7 was then filled with fluoroscein solution, and this solution was then drawn into the migration channel 5 by applying a vacuum to the buffer waste reservoir 13, with the TBE buffer solution being supplied to the other reservoirs.

The sample reservoir 7 was then emptied of the remaining fluoroscein solution and filled with sample solution, and the buffer waste reservoir 13 was filled with the TBE buffer solution. The migration channel 5, now loaded with fluorescein solution, was then aligned with the light beam, such that the linear light beam irradiated the detection region of the migration channel 5 through the windows 17 in the slit array 15.

A high- voltage protocol, as given in Table 1, was then run. In a first, purging step, the fluorescein solution was purged from the migration channel 5 into the buffer waste reservoir 13. In a second step, the fluorescent microspheres in the sample reservoir 7 were drawn into and through the migration channel 5.

The time-domain signal as detected by the optical detector 27 was then recorded for a period of 120 s; the data points being stored as text files in the data acquisition unit 29 and processed using Igor Pro 3 in the processing unit 35. In this Example, the time t = 0 was arbitrarily selected within the 600 s mobilization step. Figure 5 illustrates part of the time-domain signal.

In preferred embodiments analysis of the data is by way of a Fourier transform (FT) or a wavelet transform (WT). Analysis of the data using both of these techniques will be described hereinbelow.

Fourier Transform (FT) Analysis

Data for an even number of points bracketing the 120 s sampling period was then treated with a FFT in the forward direction to yield frequency-domain data. No apodization function was employed. The FFT algorithm yields (N/2) + 1 complex points (pairs of real F_υ, _Re and imaginary FT_υ, τ_m points) in the frequency domain for an input of N real points in the time domain.

This complex data can also be represented in terms of the magnitude F_{υ> Mag} as given in equation (1) below. F_υ, Mag ~ ((F_υ, Re) + (F_υ, R_e) ) (1)

Figure 6(a) illustrates the resulting magnitude plot in the frequency domain, with a fundamental peak having a center frequency of 7.1 Hz and an S/N ratio of 16 being determined.

The velocity of the particles u is given by equation (2) below, where /is the frequency of the fundamental peak and d is the center-to-center spacing between adjacent detection windows 17.

u =fd (2)

Thus, a fundamental peak frequency of 7.1 Hz translates to a velocity of 497 μms^"1 (7.1 Hz x 70 μm).

The number of particles detected is represented by the amplitude of the fundamental peak. Thus, the frequency-domain signal also provides for measurement of numbers of particles.

Figure 6(b) illustrates in enlarged scale the section of the magnitude plot of Figure 6(a) encompassing the fundamental peak at 7.1 Hz. As will be seen, the fundamental peak comprises a plurality of narrow peaks centred about a frequency of 7.1 Hz, each peak representing a particular velocity. A plurality of peaks are observed as a plurality of particles having different velocities passed the detection region during the sampling period. This distribution in velocities arises as a result of a distribution in the charge-to- size ratios of the particles.

Wavelet Transform (WT) Analysis

WT is performed by moving a short piece of a waveform ('wavelet') along the time axis of the signal and expressing the goodness of fit at every location in a coefficient C(l,t). The wavelet is subsequently scaled with a scaling factor a and the process repeated providing coefficients C(α,t). This scaling is then repeated with further, higher scaling factors a to achieve a higher compression. Typical representations of a wavelet transform present the goodness of fit (the values of C(a,t)) in a two-dimensional plot, with the time on the x-axis and the scaling factor a on the y axis. Scaling factor a is thereby proportional to the frequency. The scaling can be done with factors of two (discrete wavelet transform) or in a continuous fashion (continuous wavelet transform). One advantage of WT over windowed FT is that WT decreases the size of the analysis window with increasing frequency, yielding a higher time resolution.

The wavelet of equation (3) was used for the determination of particle velocity.

y = exp(-x³²/2)cos(l 60x) (3)

The wavelet was designed to give an optimal balance between time and frequency resolution for the acquired data. The high power of x was chosen such as to cause the envelope of the wavelet to have steep boundaries and thereby provide a sharper resolution of the particles on the time scale. The coefficient in the cosine function was adjusted to produce a wavelet of about 50 periods, fitting exactly on the 50 emission peaks generated by a moving particle. The selection of 50 oscillations maximises the frequency information and provides a time separation between successive particles.

Figures 7(a) and (b) illustrate respectively the time-domain signal and a plot of the fit coefficients of a continuous WT with the wavelet of equation (3) for frequencies around 7 Hz. Maxima can be clearly distinguished, indicating particles travelling with particular velocities at different times. The range of frequencies observed is between 6.8 and 7.3 Hz which is in accordance with the results of the FT transform.

The velocity of the particles is derived from a multiplication of the frequency /with the center-to-center spacing d of adjacent windows 17, in this embodiment 70 μm, as given above in equation (2).

The number of particles detected is determined from a determination of the number of maxima. In this Example, from a visual determination of the plot of the fit coefficients, the number of maxima is estimated at between 60 and 70 particles, which is in accordance with the number calculated from the particle concentration.

Finally, it will be understood that the present invention has been described in its preferred embodiments and can be modified in many different ways without departing from the scope of the invention as defined by the appended claims.

In one alternative embodiment the slit array 15 could be formed such that the windows 17 have a width which is half the center-to-center spacing of adjacent windows 17, for example, windows 17 having a width of 35 μm and a center-to-center spacing of 70 μm. In this way, the first harmonics could be removed from the frequency-domain plots.

Also, in the described embodiment, particles are electrophoretically driven tlirough a fluid medium. It should be appreciated that this transport mechanism is exemplary and that many transport mechanisms could be employed. For example, particles could be transported in a fluid flow through the migration channel or driven through a fluid medium in the migration channel. In particular, particles could be pressure driven through the migration channel, driven through the migration channel by hydrodynamic chromatography, driven through the migration channel by flow-field fractionation, driven through the migration channel by electrophoresis, in particular dielectrophoresis, and driven through the migration channel by hydrodynamic pumping, in particular electrohydrodynamic pumping and magnetohydrodynamic pumping.

Further, in the described embodiment, the detection of particles is by the fluorescent emission of the particles. It should be appreciated that this particular kind of optical detection is exemplary and that many detection techniques could be utilized. Other techniques include detection of the light scattering from the particles and the optical absorption by the particles.

Still further, in the described embodiment, the Shah detection function is achieved by utilising a slit array 15 to illuminate a plurality of spaced sections in the detection region of the migration channel 5. It should be appreciated that this illumination technique is exemplary and that the Shah detection function could be otherwise achieved. For example, a plurality of spaced light elements could be utilized to illuminate the plurality of spaced sections in the detection region of the migration channel 5. In one embodiment the measurement system could comprise at least one light source and a plurality of light-transmitting elements, such as holographic lenses, waveguides and optical fibers, coupled to the at least one light source and disposed in spaced relation to the detection region in the migration channel 5 such as to illuminate respective ones of the plurality of spaced sections in the detection region of the migration channel 5. In an alternative embodiment, the Shah detection function could be realized through the detection technique instead of the illumination technique. For example, in one embodiment the measurement system could comprise a single light source 19 for illuminating the detection region of the migration channel 5, and, as illustrated in Figure 8, an optical detector 27 which comprises a plurality of spaced detector elements 37, such as photodetectors, for detecting optical emission from a plurality of spaced sections in the detection region of the migration channel 5. In one embodiment such an optical detector 27 could be provided by a continuous array of detector elements 37, ones 37a of which are operative to detect optical emission and others 37b of which are inoperative. One advantage of such a detector 27 would be to enable the center-to- center spacing of the spaced sections in the detection region which are to be detected to be readily altered according to a required detection regime.

Still yet further, for the purposes of exemplification, the operation of the described embodiment has been described in relation to detecting fluorescent microspheres. It should be appreciated that the detection of microspheres is purely exemplary, and that the measurement technique of the present invention extends to the detection of any particles. In this regard, the measurement technique of the present invention finds particular application in the detection of single molecules, particles of particle-based assays, in particular an immunoassays, and biological cells, such as red and white blood cells, viruses and bacteria.

Claims

1. A measurement system for characterizing particles of a fluid sample, comprising: a microfluidic chip including a migration channel through which particles of a fluid sample are driven in a fluid medium; an illumination unit for illuminating at least a plurality of spaced sections in a detection region of the migration channel; an optical detector for detecting optical emission from at least the plurality of spaced sections in the detection region of the migration channel; an acquisition unit for generating a time-domain signal from the optical emission as detected; and a processing unit operably coupled to the acquisition unit for receiving the time- domain signal and transforming the time-domain signal to a frequency-domain signal, which frequency-domain signal provides for characterization of the particles.

2. The system of claim 1, wherein the system provides for measurement of velocities of particles in a fluid medium.

3. The system of claim 1 or 2, wherein the system provides for measurement of numbers of particles.

4. The system of any of claims 1 to 3, wherein the particles are transported in a fluid flow through the migration channel.

5. The system of any of claims 1 to 3, wherein the particles are driven through a fluid medium in the migration channel.

6. The system of any of claims 1 to 3, wherein the particles are pressure driven through the migration channel.

7. The system of any of claims 1 to 3, wherein the particles are driven through the migration channel by hydrodynamic chromatography.

8. The system of any of claims 1 to 3, wherein the particles are driven through the migration channel by flow-field fractionation.

9. The system of any of claims 1 to 3, wherein the particles are driven through the migration channel by electrophoresis.

10. The system of claim 9, wherein the electrophoresis is dielectrophoresis.

11. The system of any of claims 1 to 3, wherein the particles are driven through the migration channel by hydrodynamic pumping.

12. The system of claim 11, wherein the hydrodynamic pumping is electrohydrodynamic pumping.

13. The system of claim 11, wherein the hydrodynamic pumping is magnetohydrodynamic pumping.

14. The system of any of claims 1 to 13, wherein the optical detector is a fluorescence detector for detecting fluorescence of the particles.

15. The system of any of claims 1 to 13, wherein the optical detector is a light- scattering detector for detecting light scattering from the particles.

16. The system of any of claims 1 to 13, wherein the optical detector is an absorption detector for detecting optical absorption by the particles.

17. The system of any of claims 1 to 16, wherein the illumination unit includes a slit array comprising a plurality of spaced windows disposed in registration with the plurality of spaced sections in the detection region of the migration channel, and a light source for providing a light beam directed onto the slit array.

18. The system of claim 17, wherein the slit array is integrated with the microfluidic chip.

19. The system of claim 17 or 18, wherein the light source is a laser.

20. The system of any of claims 1 to 16, wherein the illumination unit comprises a plurality of spaced light elements for illuminating the plurality of spaced sections in the detection region of the migration channel.

21. The system of claim 20, wherein the illumination unit comprises at least one light source and a plurality of light-transmitting elements coupled to the at least one light source and disposed in spaced relation to the detection region in the migration channel such as to illuminate respective ones of the plurality of spaced sections in the detection region of the migration channel.

22. The system of claim 21, wherein the light-transmitting elements are integrated with the microfluidic chip.

23. The system of claim 21 or 22, wherein the light-transmitting elements comprise holographic lenses.

24. The system of any of claims 21 to 23, wherein the light-transmitting elements comprise waveguides.

25. The system of any of claims 21 to 24, wherein the light-transmitting elements comprise optical fibers.

26. The system of any of claims 21 to 25, wherein the at least one light source is a laser.

27. The system of any of claims 21 to 26, comprising a plurality of light sources, each coupled to a respective one of the light-transmitting elements.

28. The system of any of claims 1 to 27, wherein the optical detector comprises a single detector for detecting optical emission from each of the plurality of spaced sections in the detection region of the migration channel.

29. The system of any of claims 1 to 27, wherein the optical detector comprises a plurality of spaced detector elements for detecting optical emission from respective ones of the plurality of spaced sections in the detection region of the migration channel.

30. The system of claim 29, wherein the detector elements comprise photodetectors.

31. The system of any of claims 1 to 16, wherein the illumination unit comprises a single light source for illuminating the detection region of the migration channel, and the optical detector comprises a plurality of spaced detector elements for detecting optical emission from a plurality of spaced sections in the detection region of the migration channel.

32. The system of claim 31 , wherein the light source is a laser.

33. The system of claim 31 or 32, wherein the detector elements comprise photodetectors.

34. The system of any of claims 1 to 33, further comprising: a dilution stage for performing dilution of the fluid sample such that the concentration of particles is less than a predetermined concentration.

35. The system of claim 34, wherein the dilution stage is configured to provide that the concentration of particles is such that the average spacing of particles driven through the migration channel is not less than the center-to-center spacing of adjacent ones of the plurality of spaced sections in the detection region of the migration channel.

36. The system of claim 34 or 35, wherein the dilution stage is configured to provide that the concentration of particles is such that the average spacing of particles driven through the migration channel is not less than about 0.5 mm.

37. The system of claim 36, wherein the dilution stage is configured to provide that the concentration of particles is such that the average spacing of particles driven through the migration channel is not less than about 1 mm.

38. The system of any of claims 34 to 37, wherein the dilution stage is operably controlled by the processing unit such as to provide for the necessary dilution of the fluid sample.

39. The system of any of claims 1 to 38, wherein the transform is a Fourier transform.

40. The system of any of claims 1 to 38, wherein the transform is a wavelet transform.

41. The system of claim 40, wherein the transform is a continuous wavelet transform.

42. The system of any of claims 1 to 41, wherein the particles comprise single molecules.

43. The system of any of claims 1 to 41, wherein the particles comprise particles of a particle-based assay.

44. The system of claim 43, wherein the assay is an immunoassay.

45. The system of any of claims 1 to 41, wherein the particles comprise biological cells.

46. The system of claim 45, wherein the particles comprise blood cells.

47. The system of claim 45, wherein the particles comprise viruses.

48. The system of claim 45, wherein the particles comprise bacteria.

49. A method of characterizing particles of a fluid sample, comprising the steps of: driving particles of a fluid sample through a migration channel of a microfluidic chip; illuminating at least a plurality of spaced sections in a detection region of the migration channel; detecting optical emission from at least the plurality of spaced sections in the detection region of the migration channel; generating a time-domain signal from the optical emission as detected; and transforming the time-domain signal to a frequency-domain signal, which frequency-domain signal provides for characterization of the particles.

50. The method of claim 49, wherein the characterization provides for measurement of velocities of particles in a fluid medium.

51. The method of claim 49 or 50, wherein the characterization provides for measurement of numbers of particles.

52. The method of any of claims 49 to 51, wherein the particles are transported in a fluid flow through the migration channel.

53. The method of any of claims 49 to 51 , wherein the particles are driven through a fluid medium in the migration channel.

54. The method of any of claims 49 to 51, wherein the particles are pressure driven through the migration channel.

55. The method of any of claims 49 to 51, wherein the particles are driven through the migration channel by hydrodynamic chromatography.

56. The method of any of claims 49 to 51, wherein the particles are driven through the migration channel by flow-field fractionation.

57. The method of any of claims 49 to 51, wherein the particles are driven through the migration channel by electrophoresis.

58. The method of claim 57, wherein the electrophoresis is dielectrophoresis.

59. The method of any of claims 49 to 51, wherein the particles are driven through the migration channel by hydrodynamic pumping.

60. The method of claim 59, wherein the hydrodynamic pumping is electrohydrodynamic pumping.

61. The method of claim 59, wherein the hydrodynamic pumping is magnetohydrodynamic pumping.

62. The method of any of claims 49 to 61, wherein the step of detecting optical emission comprises the step of detecting fluorescence of the particles.

63. The method of any of claims 49 to 61, wherein the step of detecting optical emission comprises the step of detecting light scattering from the particles.

64. The method of any of claims 49 to 61, wherein the step of detecting optical emission comprises the step of detecting optical absorption by the particles.

65. The method of any of claims 49 to 64, wherein the step of illuminating at least a plurality of spaced sections in the detection region of the migration channel comprises the step of illuminating a plurality of spaced sections in the detection region of the migration channel.

66. The method of claim 65, wherein illumination is from a laser.

67. The method of any of claims 49 to 66, wherein the step of detecting optical emission comprises the step of detecting optical emission from each of the plurality of spaced sections in the detection region of the migration channel using a single detector.

68. The method of any of claims 49 to 66, wherein the step of detecting optical emission comprises the step of detecting optical emission from the plurality of spaced sections in the detection region of the migration channel using a plurality of spaced detector elements, each detecting optical emission from a respective one of the plurality of spaced sections in the detection region of the migration channel.

69. The method of any of claims 49 to 64, wherein the step of illuminating at least a plurality of spaced sections in the detection region of the migration channel comprises the step of illuminating the detection region of the migration channel, and the step of detecting optical emission comprises the step of detecting optical emission from a plurality of spaced sections in the detection region of the migration channel using a plurality of spaced detector elements, each detecting optical emission from a respective one of the plurality of spaced sections in the detection region of the migration channel.

70. The method of claim 69, wherein illumination is from a laser.

71. The method of any of claims 49 to 70, further comprising the step of: diluting the concentration of particles in the fluid sample such that the concentration of particles is less than a predetermined concentration.

72. The method of claim 71, wherein the concentration of particles is such that the average spacing of particles driven through the migration channel is not less than the center-to-center spacing of adjacent ones of the plurality of spaced sections in the detection region of the migration channel.

73. The method of claim 71 or 72, wherein the concentration of particles is such that the average spacing of particles driven through the migration channel is not less than about 0.5 mm.

74. The method of claim 73, wherein the concentration of particles is such that the average spacing of particles driven through the migration channel is not less than about 1 mm.

75. The method of any of claims 71 to 74, wherein the dilution of the fluid sample is in response to the generated time-domain signal.

76. The method of any of claims 49 to 75, wherein the transform is a Fourier transform.

77. The method of any of claims 49 to 75, wherein the transform is a wavelet transform.

78. The method of claim 77, wherein the transform is a continuous wavelet transform.

79. The method of any of claims 49 to 78, wherein the particles comprise single molecules.

80. The method of any of claims 49 to 78, wherein the particles comprise particles of a particle-based assay.

81. The method of claim 80, wherein the assay is an immunoassay.

82. The method of any of claims 49 to 78, wherein the particles comprise biological cells.

83. The method of claim 82, wherein the particles comprise blood cells.

84. The method of claim 82, wherein the particles comprise viruses.

85. The method of claim 82, wherein the particles comprise bacteria.

86. The method of any of claims 49 to 85, wherein the fluid sample comprises a single fluid sample including a plurality of particles of different kind, whereby the method provides for characterization of each different kind of particle.