EP1977229A1

EP1977229A1 - Acoustic sensor sperm test kit and assay

Info

Publication number: EP1977229A1
Application number: EP07705031A
Authority: EP
Inventors: David C. Hughes; Glen Mchale; Michael Ian Newton
Original assignee: Nottingham Trent University
Current assignee: Nottingham Trent University
Priority date: 2006-01-25
Filing date: 2007-01-24
Publication date: 2008-10-08
Also published as: GB0601492D0; CA2640114A1; WO2007085839A1

Abstract

The invention relates to a device for assaying sperm motility comprising: (a) a sample inlet port (32); (b) a sperm detector comprising: a first sample flow channel (38) extending from the inlet port (32); and a sperm detection region (34) in fluid communication with said flow channel (38); wherein the sperm detection region comprises an acoustic wave sensor, or microfabricated cantilever (50), and a sperm binding surface (44) capable of binding sperm. Methods of assaying for sperm motility are also provided comprising: (i) Providing a sample of sperm at an inlet; and (ii) Determining the rate at which sperm bind to a sperm detector distal to the inlet.

Description

ACOUSTIC SENSOR SPERM TEST KIT AND ASSAY

The invention relates to assays and kits for detection of sperm quality.

Reduced fertility is a significant health care problem, with 1 in 6 couples in the UK experiencing difficulties in conceiving. Frequently the cause is reduced fertility in the male partner. According to the World Health Organization (WHO) criteria for male fertility, the primary factors governing male fertility are semen volume, sperm cell density, motility, forward progression, and morphology. The WHO guidance for 'normality' is 10 million motile sperm per millilitre. Currently, nearly all semen samples are analyzed in the laboratory and a trained technician is required to perform semen analysis predominantly by direct observation under a microscope. The sample is observed and several aspects of the sperm cell composition are quantified, such as density, percent motility, percent morphologically abnormal, and forward progression. Whilst some automation techniques have been applied using modern image processing and analysis software, the majority of samples analysed still rely on direct human observation. Recently, single use, over-the-counter products that perform some basic semen analysis are becoming available.

Colourimetric assays for testing for semen quality are known in the art. For example, Genosis Ltd. (Kingston upon Thames, England) have produced a product sold under the trademark "Fertell". Semen is collected and mixed with a buffer. The solution containing sperm, where present, is heated to 37⁰C and the sperm allowed to swim up a capillary channel to a test area. The presence of sperm is detected with anti-CD59 monoclonal antibody conjugated with colloidal gold. Sperm bound to the antibody is trapped on a nitrocellulose strip. Unreacted conjugate is washed from the strip with excess buffer.

WO 93/22053 also shows a colourimetric assay using microchannels through which a variety of analytes, such as sperm passes to a detection region utilising antibodies to detect the sperm. The application also speculates that pressure detectors, conductivity and restricted flow might be used to detect the analyte. A colourimetric test kit is also shown in US 5,434,027.

A problem with such colourimetric devices is that they usually produce a yes/no answer if the presence of sperm is, for example, after a predetermined time threshold. They show that the sperm is motile, but do not give an indication of the quality of the sperm or an accurate quantitative determination over time.

US 2004/0146850A and US 2005/301322 disclose devices utilising a microchannel through which sperm swim to a detector. Sperm is preferably detected using a fluorescent reporter having a cleavable ester group that promotes uptake of a reporter molecule into sperm in an uncharged state and inhibits efflux of the reporter from sperm in a charged cleaved coloured state. The fluorescently labelled sperm may then be detected. Alternatively, magnetic or conductive metal particles may be used to label the sperm. One potential problem is that the label itself may have an effect on the sperm.

US 5,427,946 and US 5,744,366 disclose using a variety of methods including complex optical systems, such as a camera, to detect sperm swimming through a microchannel to an egg.

Photometric methods of detecting using a number of distinct pathways of light through a sample chamber is disclosed in US 6,426,213. Optical devices, utilising a microscope to count the sperm cells are disclosed in EP 0437408.

Optical assays utilising the absorbance of a sample at e.g. 400 to 700 nm have also been used to determine sperm density in a sample (US 4,632,562). More complex devices for determining sperm motility by looking at variations in optical density due to the motion of cells is disclosed in US 4,176,953.

The inventors have realised that there is a need to produce more quantitative assays that are readily incorporated into portable devices for use in general practice, veterinary surgeries, on farms or in the home. The ability to quantatively determine not only the presence of motile sperm, but an indication of the numbers and rate of motility allows the effect of environmental factors to be monitored. For example, nutrition, smoking and the effects of tight fitting underwear have previously been observed to affect sperm motility. The ability to monitor sperm quality as it is affected by changes in e.g. diet, stopping smoking, or changes in clothing provides an alternative way of a couple improving their chances of having a baby without resorting to surgical alternatives. Additionally such a device would be able to be used for veterinary uses, such as testing sperm quality of stud animals and in particular as a field instrument for use immediately prior to artificial insemination.

The inventors have utilised acoustic wave-type devices and a time of flight techniques to detect semen that have passed through a channel.

Acoustic wave sensors are based on the ability of acoustic wave devices to detect very small changes in mass attached to their surface and usually contain a sensitizing layer that can recognize and bind the 'species' we want to detect onto the mass sensitive surface. Although not the only way of producing acoustic waves, the piezoelectric effect is the main technique used in sensor applications; a voltage applied to a piezoelectric material will result in mechanical stress. The most widely used acoustic wave device for sensor applications is the thickness shear mode device (TSM) or quartz crystal rnicrobalance (QCM). A quartz crystal, 101, is cut in a thin slab and placed between two metal electrodes, 101, 102 (figure Ia). The straight and forked arrows shown at 102 indicates that the surface oscillates between the two positions indicated by the arrows. If an ac voltage is applied between the two plates, the crystal starts to oscillate with transverse (shear) waves generated travelling across the thickness of the crystal, the displacement represented by 104. The crystals may be incorporated in oscillator circuits that produce an ac voltage at the resonant frequency of the crystal. Oscillators like this work from 50kHz up to 20MHz and form the basis of quartz watches which can provide a stability of 1 part in 10¹⁰ or a drift of 1 second in 300 years. The oscillation becomes very large when the oscillating frequency corresponds to matching the thickness of the crystal with a whole number of half wavelengths so for plate thickness d and wavelength λ and where n is an integer: λ = 2d/n. For AT cut quartz crystals this corresponds to a thickness of d = 1.67/ f where f is measured in MHz and d in mm so for a 5MHz crystal operated with n=l (fundamental) corresponds to a thickness of 0.334mm. The Sauerbrey equation (1957) relates the change of the crystals resonant frequency is proportional to the change in rigid mass on the crystal surface; for AT cut quartz this gives Δf =-2.26x10^"6U²Am/ A where Δf (in Hz) is the change in frequency that occurs for an increase in mass Δm (in grams) on the surface of area A (in cm²) with a crystal resonant frequency of f (in Hz); the constant comes from the crystal materials properties. A well-designed oscillator circuit can still resonate a quartz crystal with one or both, faces immersed in a liquid. The change in mass rigidly attached to the surface still causes a proportional change in frequency although changes in other parameters such as the liquids viscosity (η) and density (p) will also cause changes in frequency. The acoustic wave will only sense changes within a short distance into the liquid called the penetration depth. Non-rigid attached mass and liquid density and viscosity changes also produce damping of the crystal oscillations and so can be used as sensor responses.

A surface acoustic wave (SAW) is a combined mechanical and electromagnetic field disturbance that is localised to around one wavelength of the surface of a solid. On a piezoelectric substrate a SAW can be generated by lithographically fabricating a set of metal interdigital transducers (E)T) and applying a radio frequency voltage across them. The spacing of the fingers of the IDT determines the wavelength and this together with the substrate orientation, which defines the propagation speed, determines the frequency of operation. SAW devices are used as the frequency control element in communications devices (mobile phones, etc). A SAW delay line device provides an area between the IDT's that can be used as a sensor. As the wave is surface localised, any rigid mass deposited from the vapour phase onto the propagation path changes the speed of propagation and hence the resonant frequency of the device. The downward shift in the frequency response can be made entirely dependent on surface mass loading by metallising the propagation path and shorting the electric field boundary conditions. For a vapour phase mass response, the shift in frequency of a SAW device is proportional to the square of the frequency, so that fabricating IDT' s with smaller wavelengths provides enhanced sensitivity.

In Rayleigh SAW devices, the surface normal component of oscillation leads to large attenuation in liquid environments. However, other modes, such as, shear horizontally polarised surface acoustic waves (SH-SAWs), surface skimming bulk waves (SSBW), surface transverse waves (STW), acoustic plate modes (APM) (Figure Ib) and Love waves (Figure Ic) have only in-plane components of surface oscillation. These show input transducers 105 and output transducers 106, together with a waveguide layer 107. Such modes are also excited using lithographically fabricated IDT 's. In these modes, the motion of the liquid entrained by the oscillation is damped and decays within a penetration depth δ = V(2η/ωp) of the interface, where ω is the angular frequency, in the same way as the thickness shear mode device. The liquid entrainment is shown schematically in fig. 2 although the size of the penetration depth is much exaggerated; in water for a 150 MHz oscillation d ~40 nm. At its simplest level, the frequency response of the acoustic shear wave oscillation measures the interfacial mass within the penetration depth. A more complex measurement approach for example using a network analyser to look at changes in both insertion loss and phase can help to extract information on conformational changes of attached layers. Over the last decade, this concept has provided a powerful new technique for monitoring solid-liquid interfacial changes with applications including in-situ monitoring of electrochemically induced changes in films and chemical and biological sensing. Most recently, it has been realised that acoustic wave devices can respond, not only to surface deposited mass, but also to the viscoelastic properties of (acoustically thick) films. The combination of frequency and dissipation indicates the viscoelastic nature of polymer films and it is therefore possible to extract quantitative information on the polymer's high frequency shear modulii.

Acoustic wave devices are sensitive to environmental parameters such as temperature and pressure. To reduce this effect to a usable level the sensor/reference technique is often employed. Here two acoustic wave sensors are used each in an oscillator configuration. The only difference between the two is that one will be coated with the sensitising layer and the other not. By using the difference between the two frequencies rather than the absolute frequency, changes which are mostly due to the binding of the target are measured and drift due to environmental parameters can be made negligible.

GB 2 371 362 A shows an example of one use of a SAW detector, for detecting particles in a gas stream.

A typical human sperm, for example, has a head diameter of approximately 5 μm and a total length of 50-60 μm with a mass of between 15-37 pg. The detection of a single sperm is below the detection level for some acoustic wave sensors however the assaying requires the detection of many thousands of sperm produced in a typical semen sample.

The invention provides a device for assaying sperm motility comprising:

(a) a sample inlet port;

(b) a sperm detector comprising: a first sample flow channel extending from said inlet port; and a sperm detection region in fluid communication with said flow channel, characterised that said sperm detection region comprises an acoustic wave sensor, or microfabricated cantilever, and a sperm binding surface capable of binding to sperm.

The ability to use such devices allows, for example, microfabricated chips to be produced very cost-efficiently that can be simply disposed of once an assay has been carried out. Chips may be simply plugged into the necessary recordal equipment, used and then disposed of. The sperm detection region is distal to the inlet port to allow a 'time of flight¹ measurement of sperm swimming from the inlet port, through the channel, to the sperm detection region. As previously indicated, such devices may be improved by putting a reference chamber in which takes into account environmental changes such as changes in temperature and pressure which might affect the sensitivity of the acoustic wave sensor. Accordingly, preferably the device additionally comprises:

(c) a reference detector; a second sample flow channel extending from said inlet port; and a reference detection region, said reference detection region comprising an acoustic wave sensor, or microfabricated cantilever, and a surface to which sperm does not substantially bind.

Acoustic wave devices per se are known in the art, although not in combination with the currently claimed invention. Preferably, the acoustic wave device is selected from a surface acoustic wave sensor, a quartz crystal microbalance, an acoustic plate mode sensor, a flexible plate mode sensor, a shear horizontally polarised surface acoustic wave sensor, a surface transverse wave sensor, layer guided acoustic plate mode sensor, and a Love wave sensor; acoustic wave sensors are reviewed in Ballentine et al [Acoustic Wave Sensors: Theory, Design, and Physico-Chemical Applications (Applications of Modern Acoustics) David S. Ballantine, Robert Marshall White, SJ. Martin, Antonio J. Ricco, E. T. Zellers, G. C. Frye, H. Wohltjen, Moises Levy (Editor), Richard Stern (Editor)] In addition microfabricated cantilevers may also be employed [e.g. A chemical sensor based on a microfabricated cantilever array with simultaneous resonance- frequency and bending readout F. M. Battiston, J.-P. Ramseyer, H. P. Lang, M. K. Bailer, Ch. Gerber, J. K. Gimzewski, E. Meyer, H.-J. Gϋntherodt Sens. Actuators B 77, 122 (2001)]. Microfabricated cantilevers work in a similar manner to acoustic wave devices in that they change their resonant frequency as a result of mass attachment to the surface

The flow channel may have a diameter of a few μm, which makes it particularly applicable to microfabrication, up to potentially any diameter. Indeed, the flow channel may be substantially the same diameter as, for example, the inlet port and sperm detection region. Indeed, the inlet port and/or the sperm detection region may form a part of the proximal end and the distal end of the channel respectively. Alternatively, separate chambers connected via the flow channel may be provided for one or more of the inlet port or the sensor region. Additionally, the reference region may also be formed integrally with the flow channel, or alternatively may be provided in flow communication with the inlet port, for example via a second sample flow channel.

Given a typical straight line velocity for sperm of 20-60μm per second, the length of the flow channel may be set to give the time at which the first of the sperm arrive at the detector for example a channel length of 60 mm would equate to the first sperm arriving after 16 minutes. The use of a channel spreads the arrival time of the sperm at the detection region out, providing more information on the ability of the sperm to swim.

The sperm binding surface capable of binding sperm may be any compound to which sperm are known to bind, including poly-L-lysine, or an antibody against one or more substances in the sperm, such as anti-CD59 antibodies or other antibodies known in the art. Methods of attaching antibodies to supports are themselves well known in the art.

The assay can be further improved by identifying whether the sperm is capable of binding to an egg or one of its components. One of the causes of infertility is the inability of sperm to bind to an egg. Eggs are surrounded by a space, called the perivitilline space. Surrounding this space is an extracellular matrix, called the zona pellucida. Sperm must be able to bind to the zona pellucida prior to fertilising the egg. In mammals, on sperm contact substances are liberated into the perivitilline space which modify the zona pellucida resulting in a block to further sperm penetration.

The surface may therefore be a portion of zona pellucida (for example, without an egg). The human zona pellucida is comprised of four glycoproteins (ZPl, ZP2, ZP3 and ZP4) (Lefievre et al Human Reprod. 2004, Conner et al Human Reprod. 2005). There is evidence to suggest that human sperm initially bind to a complex of ZP3 and ZP4. Acrosome reacted sperm then bind to ZP2. Such proteins may be obtained from natural sources or be recombinantly produced using techniques known in the art.

Additionally, other sperm binding proteins associated with the cervix may be used. For example, hyaluronan is found in the mucus of the cervix and is known to bind sperm and increase the tail motion of sperm. This has been suggested as a coating for determining sperm interaction (Johnston J.B. and Huszar G., technical paper Biocoat Incorporation, Horsham PA. www.biocoat.com/spermattech.asp).

Preferably two or more different sperm detection regions are provided, each with a different sperm binding surface. This allows the interaction of the sperm with binding compounds to be assayed and problems with the ability of sperm to bind compounds to be identified in more detail.

Preferably, the second acoustic wave sensor comprises a surface coating which sperm does not substantially bind to, which may be as simple as a clean uncoated piezoelectric substrate. Other suitable surfaces could be produced using materials such as PTFE or thin oily firms.

Preferably, each sensor comprises one or more interdigitated transducer resonators to allow the change in the frequency observed using the acoustic wave sensor, upon binding of the sperm, to be detected.

The device of the invention may additionally comprise a heater to warm the device. For example, the device may be warmed to the body temperature of the animal from which the sperm is obtained. For example, this may be 37⁰C should human sperm be detected. Such a temperature controlled environment may actually negate the requirement for a reference device.

Preferably, the sperm detected is obtained from mammals or birds. Mammals include both human and non-human animals such as cows, pigs, horses, sheep, goats, dogs and cats. Avian sources of sperm include both turkeys and chickens. The testing of sperm of the domesticated animal is often useful to be able to identify whether, for example, a pedigree bull is fertile to allow it to be used as a source of semen for artificial insemination. Artificial insemination is regularly used for agricultural purposes with over 100 million inseminations carried out worldwide each year and it is essential that the semen used is of high quality.

The device may additionally comprise, for example, a timer and date recorder to allow recordal of the time for movement of the sperm from the inlet port to the sperm detector. This may be in the form of a personal computer to which the device may be attached. Alternatively, an integrated recorder and timer may be produced into which the device may be integrated, along with, for example, a heater. Measurement electronics for acoustic wave oscillators are well known in the literature with indicative techniques reviewed in Ballentine et al.

The invention also provides an assay kit comprising a device, according to the invention, and one or more additional items, including a buffer, a diluent, a connector for connecting the assay kit to a personal computer and instructions for using the device. Use of a suitable buffer or diluent, such as phosphate buffered saline (PBS) may be used to dilute the semen sample and allow the sperm to more freely move.

A further aspect of the invention provides a method of determining sperm quality comprising:

(i) Providing a sample of sperm at an inlet; and

(ii) Determining the rate at which sperm bind to a sperm detector distal to the inlet.

Preferably the method comprises the step of timing the time for which sperm move from the inlet to a sperm detector where the sperm bind and are preferably detected by an acoustic wave sensor or microfabricated cantilever. Preferably, this uses a device according to the invention. In particular, in a preferred method of the invention the sperm are timed moving from an inlet port, through a channel to the sperm detector.

Preferably the rate of change in the phase or the frequency of the sensor is determined.

The invention will now be described by way of example only, with reference to the following figures:

Figure 1 shows a schematic diagram of a) quartz crystal microbalance, b) acoustic plate mode device and c) Love wave device.

Figure 2 shows a schematic diagram of acoustic wave entrainment.

Figure 3 shows a schematic diagram of QCM based test rig according to the invention.

Figure 4 shows data obtained from the test rig shown in Figure 3, the frequency change of the QCM as a function of time for the reference (upper line) and sensing (lower line) crystals. The arrow shows the time at which the sperm sample was introduced to the inlet port.

Figure 5 shows a reflected power spectrum of quartz crystal with buffer only (line 1), with, sperm after 1 hour (line 2) and with excess sperm removed and fresh buffer (line 3).

Figure 6 shows a schematic diagram of an APM based test rig according to the invention.

Figure 7 shows data obtained from the test rig shown in Figure 6, the frequency change of the APM as a function of time; the arrow shows the time at which the sperm sample was introduced to the inlet port. Figure 8 shows a schematic diagram of a sensor according to the invention from above (figure 8a) and to one side (figure 8b)

Preliminary data was obtained using two different types of acoustic wave device:

Example 1 Quartz crystal microbalance

Pig semen samples were supplied by a commercial artificial insemination centre (JSR Genetics, Driffield, UK). Prior to despatch the semen is mixed with a dilutent (Androhep) cooled to 17⁰C packaged in plastic bottles, and delivered by overnight postal service. This medium is suitable for up to 5 days storage at ambient temperature. Prior to use, the sperm were allowed to settle to the bottom of the container overnight and excess dilutent removed; the remaining concentrated sperm in dilutent were then shaken every day to prevent settlement. A flow cell was fabricated to use two polished 5MHz quartz crystals with gold electrodes (Testboume 149211-1) as the sensing and reference as shown in Figure 3. Figure 3 shows a side view (a) and top view (b) with a sample inlet port 301 and flow channel 302 connecting the inlet port 301 to a sensor 303, comprising a speπn detection region 304, and a reference device 305. A bore hole 306 is provided to prevent pressure increases giving an unwanted signal.

The sensing crystal was coated with poly-L-lysine and the reference left uncoated. To prepare the poly-L-lysine coated crystals, they were initially cleaned with ethanol, then ozone treated for 30 minutes. The crystals were then placed in poly-L-lysine solution overnight; the devices were then washed in the PBS buffer to remove any excess. The blank crystals were cleaned with ethanol followed by PBS buffer. The crystals were used with two Maxtek PLOlO phase lock oscillators interfaced to a computer and data was collected 36 times a minute. A channel length was set to around 6cm to give a typical time of flight of around 10 to 15 minutes from the literature values of for speπn velocity (M.Hirai et al J. Androl. 22, 104 2001 and CHoIt et al, J.Androl. 18, 312 1997) and the swim up medium used was PBS buffer.

Figure 4 shows the change in frequency for the reference crystal (upper line) and the sensing crystal (lower line) for a period of 55 minutes; a 0.2ml semen sample was introduced at 16 minutes. The reference crystal shows a small positive drift in frequency of 5Hz over the measurement period with a small deviation as the semen was introduced. The poly-L-lysine coated crystal shows a negative drift prior to and after the sperm detection, this suggest a take up of particulate matter from the PBS buffer. This suggests that it may be appropriate to use a temperature controlled environment, possibly at elevated temperatures closer to body temperature, as an alternative to a reference device. At 12 minutes from the introduction of the semen a fall in frequency is observed which is completed after a further 8 minutes and shows a frequency decrease of 14.7±0.7 Hz. Confirmation that the signals observed are from the attachment of sperm were made by using the flow cell with uncoated and poly-L-lysine coated glass slides in place of the quartz crystals for different periods of time after introducing the sperm. The slides were checked using conventional microscopy to identify the sperm concentration present. In addition, a slide was coated with poly-L-lysine and the semen added directly on top, this gave 125 sperm for a 200x200 μm area after an hour. The same area on the poly-L-lysine coated slide at the end of the flow cell gave 17±3 sperm for both 15 minutes and 40 minutes after introducing the sperm suggesting that saturation does coincide with the end of the frequency change; this corresponds to 56000±10000 sperm attached to the active area of the QCM.

To assess if significant dissipation was taking place following the sperm attachment, a network analyser (Agilent 8712ET) was used to record the reflected power resonance peak for a poly-L-lysine coated crystal in PBS. The crystal holder was arranged to have the crystal horizontal so that gravitational settlement could occur and hence a maximum attachment of sperm could be achieved. Figure 5 shows the spectrum for the crystal in PBS buffer (line 1), for the crystal after a 0.2ml charge of semen had been added and left for an hour (line 2) and after the semen had been washed out and clean buffer included (line 3). The frequency of the peak shown on the network analyser peak is expected to track the frequency that would be achieved using the oscillator circuit however, if significant dissipation occurs i.e. a non-Sauebrey relationship hold, then it would be expected that the peak to broaden. The difference between line 1 and line 2 shows such a broadening is obtained with the gravitational settlement of the sperm onto the crystal surface. However, after surplus speπn are removed the resonance of line 3 is as shaip as line 1. hi a Butterworth - Van Dyke (BVD) equivalent circuit model for the crystal, the crystal resistance represents energy loss processes and a change in this shows departure from a rigid mass attachment model. For the initial PBS buffer R=368.4Ω one hour after introducing the sperm R=384.2Ω and after the excess have been removed and clean buffer introduced R=367.5Ω. This suggests that a simplified model based on the Sauerbrey equation and a sperm effective mass may be appropriate. Previous studies have estimated a dry head mass of 13ρg (G.P.Bahr and E.Zeitler J. Cell. Biol. 21, 175 1964) and up to 70% of the total sperm mass to be made up of water (L.B.Da Silva et al Science 258, 269 1992). Applying the Sauerbrey relationship to the data shown in figure 4 and using the estimate for the number of sperm attached, this corresponds to a pig sperm effective mass at 5MHz of 6.4±1.4pg.

Example 2: Acoustic plate mode (APM) device

Figure 6 shows the flow cell arrangement using an acoustic plate mode device as the acoustic wave sensor. Figure 6 (a) shows a side view, (b) shows a top view. A sample inlet port 601 is connected via flow channels 602 to a sperm detection region 603 comprising two IDTs. A bore hole 605 is provided to prevent pressure increases giving an unwanted signal.

The APM devices were fabricated on 36° rotated Y-cut X propagating lithium tantalate of thickness between 530-540 μm. The interdigital transducers (IDT) were of a double-double design with lOμm finger widths and spaces, 50 repeat patterns, a 2000μm aperture, a path length of 8mm giving a fundamental frequency of 52MHz. The fingers consisted of 40 nm of titam^'um followed by 200 nm of gold deposited by sputter coating. Both faces of the APM device were coated in poly-L-lysine by initially cleaning with ethanol, then ozone treated for 30 minutes. The devices were then placed in poly-L-lysine solution overnight; the devices were then washed in the PBS buffer to remove any excess. The face of the device not containing the IDT' s was used as the sperm detection region.

The APM device was used as the feedback element in an oscillator circuit consisting of three cascaded amplifiers (Minicircuits ZFL-500LN), a 50MHz high pass filter and 150MHz low pass filter (Minicircuits BHP-50 and BPL-150), a power splitter (M/ A com TlOOO) and a frequency counter (Agilent 53132A) interfaced to a microcomputer. The channel length was set to approximately 6.5 cm. Figure 7 shows the change in frequency for a period of 110 minutes; a 0.2ml pig semen sample was introduced at 31 minutes. At 19 minutes from the introduction of the semen a fall in frequency is observed which is completed after a further 23 minutes and shows a frequency decrease of 1780±160Hz. A previous report suggested that the sensitivity for a 52 MHz APM device is around 73.3Hz/ng mm^"2 (FJosse et al Sensors and Actuators A: Physical 53 243 1996). Using this sensitivity value and the frequency fall observed, the mass of sperm attached to the sensing area of the APM is 390±35ng. As the APM flow cell had only a single path, unlike the QCM flow cell, confirmation of the number of attached sperm was again estimated again using a poly-L-lysine coated glass slides in place of the APM one hour after introducing the sperm. The slides were checked using conventional microscopy to identify the sperm concentration present. An area of 0.36mm x 0.49 mm on the glass slide at the end of the flow cell gave 237±40 sperm and this equates to 21500±3600 sperm in the path between the EDT's of the APM device. Using these values and the frequency change of the APM, the effective mass of pig sperm on a 52MHz APM device is 18.9±4.8ρg.

The assays were carried out at room temperature.

Figures 8a and 8b show schematic diagrams of preferred detectors according to the invention. Figure 8a shows the device from above, and Figure 8b shows the device from one side. The device (30) comprises a sample well (32) which acts as an inlet port into which a sample is provided. The sample well may comprise a suitable buffer, such as PBS. Sperm swims from the sample well (32) to the sperm detection region (34) or the reference detection region (36) via channels (38 or 40). The sample well (32), sperm detection region (34), reference detection region (36) and channels (38 and 40) may be formed within any suitable material, such as PTFE (42).

The sperm detection region (34) comprises a layer of a substance that sperm will bind to, such as ρoly-L-lysine (44) on a layer of piezoelectric material (46). Sperm swimming from the sample well (32), through the flow channel (38) to the sperm detection region (34) are detected when they bind to the sperm binding layer (44). The reference detection region (36) provides a control region as it is has a layer that substantially does not bind sperm (48), such as a layer of PTFE or a clean piezoelectric wafer surface. Below the regions (34 and 36) are provided with IDTs (50). The binding of the sperm to the layer (44) causes a change in the observed resonant frequency which can be detected. Alternatively, the SAW device illustrated in Figure 8b may be replaced by another acoustic wave device, such as a quartz crystal microbalance or another acoustic wave sensor known in the art or a microfabricated cantilever.

Claims

1. A device for assaying sperm motility comprising:

(a) a sample inlet port;

(b) a sperm detector comprising: a first sample flow channel extending from said inlet port; and a sperm detection region in fluid communication with said flow channel; characterised that said sperm detection region comprises an acoustic wave sensor or a microfabricated cantilever, and a sperm binding surface capable of binding to sperm.

2. A device according to claim 1, additionally comprising:

3. A device according to claim 1 or claim 2, wherein the acoustic wave device is selected from a surface acoustic wave sensor, a quartz crystal microbalance, an acoustic plate mode sensor, a flexible plate mode sensor, a shear horizontally polarised surface acoustic wave sensor, a surface transverse wave sensor, layer guided acoustic plate mode sensor, Love wave sensor or microfabricated cantilever.

4. A device according to any preceding claim, wherein the device is microfabricated.

5. A device according to any preceding claim wherein the sperm binding surface comprises poly-L-lysine, anti-CD59 antibody, a portion of the zona pellucida from an egg, or a protein or polysaccharide found in the zona pellucida.

6. A device according to any one of claims 2 to 5, wherein the second acoustic wave sensor comprises a surface coating to which sperm will not readily attach.

7. A device according to any preceding claim wherein each sensor comprises one or more interdigitated transducer resonators.

8. A device according to any preceding claim, additionally comprising a heater to warm the device.

9. A device according to any preceding claim additionally comprising a timer and data recorder to allow recordal of the time for movement of sperm from the inlet port to the sperm detector.

10. An assay kit comprising a device according to any preceding claim and one or more additional items selected from a buffer, a diluent, a connector for connecting the assay kit to a personal computer and instructions for using the device.

11. A method of determining sperm quality comprising:

(i) Providing a sample of sperm at an inlet; and

12 Method according to claim 11, comprising the step of timing the time for which sperm move from the inlet to a sperm detector where the sperm bind and are detected by an acoustic wave sensor or microfabricated cantilever.

13. A method according to claim 11 or claim 12 wherein the method comprises using a device or an assay kit according to any one of claims 1 to 9.

14. A method according to any one of claims 11 to 13, wherein the sperm are timed moving from an inlet port, through a channel, to the sperm detector.