This invention relates to instruments for measuring the optical properties of samples.
Traditional instruments for measuring the optical properties of liquid samples employ sample holders such as cuvettes, and the measurements are made on the bulk properties of the liquid.
WO 2007131945 discloses a microvolume analyser employing a drophead having a surface which is adapted to receive a drop of liquid to be tested, the drophead being positioned in use relative to a source and a detector to illuminate a drop received thereon so that the drop causes an interaction in the path of the electromagnetic radiation between the source and detector. Unlike with bulk systems, the surface of the drophead is dimensioned to constrain the drop to adopt a shape which is dominated more by surface tension forces than by gravitational forces.
Liquid drops with such small volumes cause particular considerations which do not exist for bulk volume analysers. The properties of the drop are dependent on the drop shape which is in turn dictated by the volume of liquid in the drop due to the dominance of surface tension forces over gravity. As a result, any inaccuracy in the drop volume leads to inaccuracy in measurement. A particular source of variation in drop volume is a evaporation from the drop between the time when the drop is deposited on the drophead and the time when the measurement is taken. There can be a great deal of variation in this regard, particularly if there are repeated measurements or where different operators take different measurements.
There is provided an optical instrument comprising:
The optical instrument provides a different approach to the problem of positioning an optical sample in position relative to a light source. Rather than providing a bulk sample in a container which is inserted into position between a source and detector, or depositing a drop onto a drophead below a light source, the instrument provides a drop-supporting surface carried upon a housing, with the light source being carried into and out of position on a rotating cover. The cover is designed to reveal the drop-supporting surface through an aperture when in a loading position and to conceal the drop-supporting surface when in a measurement position, and furthermore, a mechanism is provided to positively engage the cover and housing into that measurement position to ensure accurate positioning of the source and droplet.
In addition, the use of a connector receiving the light source and providing communication to the inner surface of the cover, in combination with the cover being rotatable into and out of the measurement position, provides a way of isolating the droplet from ambient light below a cover without having to manipulate the drop once it has been deposited (bearing in mind that the apparatus is preferably for use with microliter sized droplets, more preferably in the range 1-5 microliter with a particularly preferred embodiment having a 2 mm diameter plinth which receives droplets of approximately 2-3 microliter).
The connector may be wholly internal to the cover, i.e. a mounting provided within the cover for a self-contained light source such as an LED, or it may be a conduit extending through the cover to enable an external light source to illuminate the drop-supporting surface through the cover.
Preferably, said mounting further permits translational movement between the cover and the housing along said axis, and the positioning mechanism is arranged to engage and hold the cover relatively closer to the housing when in said measurement position and to cause the cover to move relatively further from the housing when the cover rotates relative to the housing away from said measurement position.
This provides a way of maximising the exposure of the droplet to the light source when in the measurement position, by bringing the connector axially closer to the droplet, while moving the connector away from the droplet when rotating the cover.
Further, preferably, the positioning mechanism comprises means for biasing the cover towards the housing along said axis.
Even more preferably, the positioning mechanism further comprises complementary shaped features provided respectively on said cover and said housing, said complementary shaped features permitting the cover and housing to move closer together under the action of the biasing means when the cover is rotated relative to the housing to the measurement position, and forcing the cover and housing apart against the biasing means when the cover is rotated relative to the housing away from the measurement position.
In a preferred embodiment the complementary shaped features are a projection on one of the cover and housing and a recess on the other of the cover and housing, wherein the recess is dimensioned and positioned relative to the projection, when the cover is in the measurement position, to at least partially receive the projection, and when the cover is rotated relative to the housing away from the measurement position the projection moves out of the recess and forces the cover and housing apart.
The projection may be provided by a ball bearing mounted in and protruding slightly from one of the cover and housing and a recess provided in the other of the cover and housing, so that when the bearing and recess are aligned the cover and housing can move closer together than when they are not aligned and the bearing forces the cover and housing further apart.
In preferred embodiments, the housing and the cover are mutually shaped, in the vicinity of the drop-supporting surface and the connecter respectively, to define a chamber which encloses said drop-supporting surface with said connector being in optical communication with the chamber when the cover is in the measurement position, the chamber opening when the cover is rotated relative to the housing to the loading position to reveal the drop-supporting surface through the aperture.
This arrangement is particularly advantageous as it encloses the drop-supporting surface in a chamber, thereby isolating it for measurement. In addition to isolating it physically to avoid disturbing the droplet, the chamber is preferably defined by opaque walls so that the only light reaching the droplet is from the source attached to the connector.
The chamber is preferably sealed such that the droplet is surrounded by a relatively small air volume. This assists in reducing the evaporation both due to the atmosphere becoming saturated and the air around the droplet being still.
The volume surrounding the drop can also be sealed to stop contamination of the working environment by toxins such as dangerous medical or biological organisms.
Preferably, the chamber further includes a receptacle for a liquid volume, spaced apart from the drop-supporting surface.
When liquid is present in such a receptacle, evaporation of the liquid assists in saturating the volume of air in the chamber, which in turn reduces evaporation from the droplet.
In a preferred embodiment, the receptacle for the liquid volume comprises a moat surrounding the drop-supporting surface.
The instrument preferably further comprises a seal provided on one of the housing and the cover to seal said chamber and isolate it from the atmosphere.
Preferably, in addition to said loading aperture in said cover, a second loading aperture is provided in said cover, such that from the measurement position the cover may be rotated relative to the housing in one direction to reveal the drop-supporting surface through the loading aperture in said loading position and in another direction to reveal the drop-supporting surface through the second loading aperture in a second loading position.
Providing a pair of loading apertures in the cover, each of which is positioned to reveal the drop-supporting surface when the cover is rotated in a different direction, assists in use of the apparatus by both left- and right-handed operators, or by an operator using either left or right hands, where the other hand is occupied.
The instrument preferably further comprises a limiting mechanism provided between the housing and cover to restrict the rotation of the cover relative to the housing.
There is also provided a method of measuring an optical property of a liquid droplet, comprising the steps of:
There is also provided the use of an optical instrument as aforesaid comprising the steps of loading, rotating and measuring as described herein.
There is also provided a drophead for supporting a droplet to be analysed, comprising a drop-supporting surface for receiving a droplet, a reservoir for holding a liquid solvent, and a separating surface isolating the reservoir from the drop-supporting surface.
Preferably, the reservoir is in the form of a moat surrounding the drop-supporting surface.
Preferably, the moat is annular and the drop-supporting surface is at the centre of the annulus.
Preferably, the drop-supporting surface is a face of a raised cylinder and the separating surface is an annular surface surrounding the cylinder and is itself surrounded by the reservoir.
Preferably, the drop-supporting surface is of a dimension sized to stably support thereon a droplet of no greater than 5 microliter.
Preferably, the drop supporting surface is a face of a first quartz member.
Further, preferably, the surrounding surface is a face of a second quartz member.
Preferably the first quartz member is substantially transparent to visible radiation.
Preferably the second quartz member is substantially opaque to visible radiation.
Preferably the first quartz member is a cylinder and the second quartz member is an annular disk surrounding the cylinder.
In an alternative preferred embodiment a plurality of said drop-supporting surfaces are provided on a body such that each drop-supporting surface is surrounded by a reservoir.
Preferably, a single reservoir is provided within which a plurality of raised formations are provided with each raised formation providing a separating surface isolating the reservoir from one or more drop-supporting surfaces located on the raised formation.
In a preferred embodiment, each drop-supporting surface is provided on a separate raised formation and said raised formations are provided in an ordered array within the reservoir.
Preferably, such a drophead, in which the raised formations are provided in an ordered array, is configured as an assay plate for receiving a plurality of droplets for analysis in a plate reader.
There is also provided a method of using a drophead as aforesaid in an optical instrument comprising the steps of depositing a droplet on the or each drop-supporting surface (or a subset thereof), and adding a liquid to the reservoir to inhibit evaporation of said droplet(s) by generating an increased level of vapour saturation in the vicinity of said droplet(s).
There is also provided an optical instrument comprising a drophead as aforesaid, a source and a detector adapted to be positioned relative to the drophead to respectively illuminate and detect illumination coupled into a droplet loaded on the drop-supporting surface.
Preferably the optical instrument further comprises a sealing mechanism to provide a sealed chamber in which the reservoir and droplet are located in use when the source and detector are in a measurement configuration.
In
The instrument operates generally by loading a liquid sample droplet on the drophead assembly 22 via the aperture when the cover is in the loading position shown in
The cover is shown in more detail in
The apertures 24, 32 are dimensioned and spaced radially from the central hole 28 to overlie the drophead assembly 22 (
The connector 22 is set into a depression in the outer surface of the cover to ensure that when an optical fiber 18 (not shown in
On the inner surface (
Also visible on the inner surface are a series of three equi-angularly spaced depressions 44, 46, 48 (i.e. angularly spaced at 120 degrees from one another) which are each dimensioned to receive a ball bearing mounted on the housing as will be described further below. Also visible and described further below is a circumferential groove 50
Referring additionally now to
If the cover is rotated from the measurement position to one loading position by a 120 degree counter-clockwise rotation (this being the position shown in
If the cover is then rotated clockwise through 120 degrees one again reaches the measurement position, and a further 120 degree clockwise rotation results in a second pin 64 reaching a stop 66 at the other end of the groove 50 and the depression 44 receiving bearing 58.
When the cover is in an intermediate position between the central measurement position and either of the loading positions either 120 degrees to clockwise or 120 degrees to counter-clockwise, the bearings 54, 56, 58 are not located in any of the depressions 44, 46, 48 but instead bear against the inner surface 36 (
Referring additionally to
Referring additionally to
In
As can be seen, in the measurement position, the optical fiber 18 is positioned directly over the drophead assembly 22. The use of a set of three bearings which closely fit into three depressions when the cover reaches the measurement position results in a very precise and positive engagement of the cover into position with the optical fiber positioned directly over a drop carried on a drop supporting surface as will be shown in further detail below. The spring biasing the cover downwards results in the cover being held in this position against accidental movement and provides a small resistance against movement away from this position, so that the operator is required to positively rotate the cover to lift it over the top of the bearing surface as it starts to rotate to one or other of the measurement positions.
The inner surface 36 of the cover 16 carries a raised circular lip 84 on which an O-ring seal 86 is mounted, so that the terminal surface of the fiber 82 is contained within and at the centre of the lip 84. Thus, the o-ring 86 makes a seal with the top surface 52 of the housing when the cover drops into position due to the depressions and the bearings being in registration and due also to the downward urging of the spring as previously described.
Referring additionally to
The purpose of the liquid in the moat is to generate a more saturated atmosphere around the liquid under test and generally within the chamber 96 defined between the inner surface 36, top surface 52 and seal 86. As this chamber 96 is sealed once the cover 16 drops into place when rotated to the measurement position, and due to the small volume of the chamber 96, a vapour equilibrium is quickly established following which evaporation of the droplet is largely inhibited.
The sealed chamber can also be used to purge the samples for delivery into a sealed container to allow for safe disposal. Such a sealed chamber for sample can be used to effect control of the humidity or indeed other environmental factors such as temperature, atmospheric type for admixtures of gases or vapours for example, sterilizing UV to kill biological molecules etc.
The droplet 98 itself sits on a drop supporting surface defined by the top of a cylindrical quartz plinth 100 mounted in a black quartz disk 102. Black quartz is used as it fuses with quartz to provide an atomically bonded structure for the drophead but one that is optically differentiated from the sample head and assist the light guiding effect through the plinth between the droplet and the detector (not shown). Light shining on the droplet 98 from the fiber 80 is coupled through the quartz plinth 100 into a detector (not shown) located immediately below, or into a fiber (not shown) immediately below the plinth 100.
For a droplet whose volume is sufficiently small so that surface tension forces dominate over gravitational forces, the optical characteristics of the droplet are dependent on both the geometry of the drop and the composition of the liquid itself. For two droplets of identical volume, surface tension forces will ensure that the shapes are also identical. Thus, one can compare the optical characteristics of two drops of exactly equal volume and any differences will be due to the optical characteristics of the respective liquids, e.g. coupling efficiency, refractive index, turbidity, colour, clarity, attenuation, fluorescence, etc. Two geometrically identical drops will have unique optical fingerprints if their composition is different, and thus by illuminating the two droplets with suitable light and measuring the transmitted light tot the detector, useful analysis can be carried out.
The technique just described is dependent, however, on the shapes of the droplets being identical at the time when the measurements are taken. While it is certainly possible using normal, careful laboratory techniques and apparatus to deposit identical small volumes of liquid to the required degree of accuracy, and while this will inevitably result in droplets of identical shape and size (leaving aside any grossly different liquids with majorly different surface tension characteristics), inaccuracies can arise if the liquid in the droplet evaporates between its deposition on the plinth and the measurement taking place. In practice interruptions and other factors may cause the delay between deposition and measurement to vary widely from one measurement to another, and if no precautions exist to prevent evaporation, this can result in the introduction of significant inaccuracies.
The moat 94 and the sealing of the chamber 96 can eliminate these inaccuracies or at least render them insignificant. Once the chamber 96 is sealed, a vapour equilibrium is quickly established following which evaporation of the droplet is largely inhibited. Thus, a long delay in taking a measurement or series of measurements does not matter because the droplet volume is stabilised against evaporation due to the vapour pressure arising from the relatively large volume of liquid in the moat saturating the volume of air in the chamber.
Firstly, a pair of drophead assemblies 22A, 22B are disposed on the top surface 52 of the housing and the apertures 24, 32 are shaped and sized to allow both drophead assemblies 22A, 22B to be simultaneously revealed. This permits an operator to load both dropheads at once. As with the
Secondly, the positioning mechanism of the ball bearings and recesses has been modified in the
The apparatus of
It will be seen in
Operation of the
Instructions are sent to the interface 200 from a processor 206 having an operator interface 208 and working memory 210 as well as a disk storage area 212. It will be appreciated that the processor, operator interface, working memory and disk storage can be provided as part of a suitably programmed general purpose computer or can be provided as dedicated hardware elements with a suitable operating system controlling the interaction of the components.
Program instructions 214 are stored on disk 212, and the disk 212 also stores various data elements such as calibration data 216 and results and reports 218. The operation of the system under the program instructions 214 will now be described from the point of view of the screens and controls presented to the operator via the interface 208 when the software 214 is in operation.
The introductory screen shown in
The first menu option just gives simple text instructions to ensure the user has things connected properly. This is the most elementary set-up instructions.
The adjustment of source is one that requires some basic attention and the user is given a number of diagnostic tests to ensure that the source is operational (e.g., making sure source is switched on, removing the fiber and checking light is coming from the source etc.).
The adjustment of the spectrometer depends on the type of spectrometer being used and requires attention to some settings on the software (e.g. setting integration time etc.).
There are some simple checks on the system that can be conducted and here to ensure the best operation of source, drop apparatus and spectrometer (e.g., adjusting source controls and optimising spectrometer to ensure signal is not saturating). The software can direct the user to optimise the performance of the system. Selecting this option then gives user directions on adjustment of the system.
Calibration standards are supplied and if these are run after the system has been optimised wavelength checks and sensitivity, linearity and reproducibility checks can be run. A service basic report is stored in the Service Archive after the measurements have been recorded and this report can be printed.
When the drop instrument is ‘blanked’ a spectrum is taken of a reference material and stored in memory of the computer. This data set is an array of light intensities against wavelengths assigned by the spectrometer. The intensity of source light transmitted through the drop sample is then stored for every wavelength. The corrected sample and reference intensity (Intensity minus the dark current for each wavelength) are then used to calculate the sample absorbance according to the algorithm:—
The average pathlength through the drop depends on the volume of the drop and has been obtained from computer modelling of the system and also from experimental determinations based on measurements. The pathlength is determined from the software based on the volume selected. The bottom half of the screen will contain a spectra along with other relevant data for each application.
In regard to the spectrum
The calibration of the drop spectrometer is based on using a commercial standard Starna Green Calibration Fluid. Other products could be used with known spectral features and peaks with known absorbances for a given measured concentration. The absorbance values at either two or three wavelengths are returned from a measurement of the standard and compared with the known values. Replicate readings are taken and averaged and standard deviations obtained. The comparison of the standard with the known value gives a calibration of the photometric accuracy of the instrument; the standard deviation is the measure of photometric reproducibility. The analysis of the results is returned automatically in a spreadsheet as shown in the following table.
The replicate number can be selected and it is advised that 10 be used as a minimum by more than 32 would be suggested based on improving the statistical validity of the tests.
The calibration tests with the drop instrument should be better than 2% accuracy and diagnostics are suggested in the software if the calibration is out to improve the measurements in a repeat calibration.
The report of the tests is filed automatically in the calibration reports file that is automatically created on the PC of the user. This report can be printed as a hard copy from the screen or from the file.
This option allows the parameters of the instrument to be automatically optimised based on the calibration results.
Referring back to the main tabs in
The “Applications” tab requires the user to select the (S) single or (D) double drop operation.
The operations are really almost the same but with deposition of two drops (sample and reference) in the latter option. The algorithms are ones that are implemented after the data acquisition to deliver results that comply with both the accepted computational methodology for these tests and complies with the accepted statistical analysis for the assay.
Taking the single drop operation as an example, the user has the following options:
1. Direct Measurement Using Calibration Graph
The standard approach to measurements in chemistry and biology is to generate a calibration graph of Absorbance against Concentration for the measurement of a dissolved component in a solution of water of some other solvent for a measurement at some selected wavelength. This Beer-Lambert calibration graph is then used to determine the concentrations of unknown solutions whose measured concentration is graphically determined from this calibration graph.
An example is given in
The software offers the user the opportunity to automatically log the data without reference to any software skills with results automatically entered into the table below. The samples measured also require replicate measurements and from the advanced error analysis the values of both concentration and concentration error are returned. Checks are made immediately on the statistical acceptability of the results as the measurements proceed (see the Table below) and a tick appears after the measurements on those sets that are statistically acceptable with options offered to allow the user to repeat the calibration measurement. A tick appears in the box if the statistics show the result is acceptable. Furthermore, suggestions as to why the calibration measurements may not have been acceptable will be given without these being requested by the user who will be prompted to repeat rather than proceed with a statistically invalid result.
A results screen may also be presented which informs the user to obtain measurements of concentration between the LOQ and LOL. The data shown in
From this graph the equations for the error-band is computed for the 3σ-error bars by the two lines displaced at an intercept on the A-axis by this range value. In the graph shown the range is 3σblank=0.0125. Hence the equations for the error band is two lines Atop=0.0126c+0.0027 (obtained from [0.0125-0.0098]) and the bottom line Abottom=0.0126c−0.0223 (obtained from [−0.0125-0.0098]). The concentration measurement is now easily computed.
The measured absorbance of the unknown is Aunknown=0.464±0.015 (3σ-value taken as error) giving the absorbance range of values AT=0.479 to AL=0.449. The calculation for the concentration of the unknown uses the equation of the best-fit line given on the graph viz. cunknown=(0.446/0.0126)+0.0098=35.41 mg/L. The concentration error is computed from the absorbance range of the unknown measurement of concentration using the two equations for Atop and Abottom. The concentration range calculation is obtained substituting the AT=0.479 into the equation for Abottom to give 38 and AL=0.449 into the equation for Atop to give 35.66.
This gives the result 35.41±2.59 (c±Δc). Actually, the errors shown here in this example of the algorithm have been exaggerated to allow error bars on the graph to be seen and these are doubled in size, so the actual real measurement obtained with these real drop analyser set of results is 35.41±1.295 mg/L. The algorithm can be described by the flow diagram of
2. RNA/DNA
Pure DNA gives 1.8 and 2.0 for RNA. Lower values indicate the presence of protein or denatured DNA. There is second useful measure of purity ratio
These ratios are useful but the DNA concentration in ng/pi based on the absorbance measurements cDNA=(A260−A320)*50*PF where PF=pathlength factor for the drop analyser. For example for a 3 μL drop, the pathlength equals 1.184 mm determined from modelling studies and experimental testing. The experimental study with the 2 mm diameter drophead delivered an equation for PF=−0.0054VD2+0.2872VD+0.3549 where VD is drop volume in microlitres. The pathlength computation to convert the value to standard 10 mm pathlength absorption measurement is simply PF/10. All values reported in the drop spectrometer software are those that correspond to the values obtained with a standard spectrophotometer and a 10 mm cuvette.
The protein screen based on these measurements depends on the method selected for example a measurement at 280 nm has a screen as shown in
The display of the UV-visible spectrum is presented together with:—
The BCA assay requires a standard curve to be generated each time it is run before the protein (unknown) can be measured.
The measurements are conducted at the λMax of 562 nm and analysed at 750 nm.
The absorbance values are proportional to the protein concentration.
Once the curve is completed the red indicator light turns green and only with this condition showing ‘go’ can the user begin to commence measurements.
With the green light activated the calibration graph disappears and is replaced by a spectrum screen.
NOTE: In order to obtain a concentration value in μg/mL the unknown sample must fall within the limits of the standard curve and concentration determinations are obtained by linear fitting between samples. The slope m of the standard curve (calibration sensitivity) and intercept on the calibration graph (c) is determined by software with least-squares fit.
(d) The concentration is then obtained
User can select method of curve fitting required from straight-line regression; zero regression line; interpolated; and cubic-spline
The required standard curves are generated each run before a protein sample (unknown concentration) is measured. The sample is measured at 750 nm and normalised at 450 nm. The screen here is one shown above for the BCA but with the Lowry method box selected.
The required standard curves are generated each run before a protein sample (unknown concentration) is measured. The sample is measured at two wavelengths, 595 nm and normalised ay 750 nm. The screen here is one shown above for the BCA but with the Bradford method box selected.
The required standard curves are generated each run before a protein sample (unknown concentration) is measured. The sample is measured at two wavelengths. Measurement is at 546 nm. The screen here is one shown above for the BCA but with the Biuret method box selected.
(ii) Double Drop
The measurement procedures are as above but with HELP notes changed to give directions for double-drop deposition.
Referring to
In similar manner to the single annular reservoir on drophead 22, the reservoir 304 may be filled with a liquid such as water or another solvent, and individual droplets deposited manually or using conventional robotic deposition systems on the individual drop-supporting surfaces 308, with the islands separating the reservoir from the drop-supporting surfaces. The liquid in the reservoir 304 provides an increased vapour pressure above the drophead 300 to prevent evaporation of the droplets during handling and reading. The drophead can be measured in a conventional microplate reader (not shown) by shining light through each droplet so that the liquid under test in the droplets interacts with the light and a detector or detector array under the plate detects light passing through each droplet for analysis.
As seen in
The invention is not limited to the embodiments described herein which may be modified without departing from the scope of the claimed invention.
Number | Date | Country | Kind |
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11162343 | Apr 2011 | EP | regional |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/EP2012/056835 | 4/13/2012 | WO | 00 | 11/20/2013 |
Publishing Document | Publishing Date | Country | Kind |
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WO2012/140232 | 10/18/2012 | WO | A |
Number | Name | Date | Kind |
---|---|---|---|
7396509 | Burns | Jul 2008 | B2 |
7964413 | Macioszek et al. | Jun 2011 | B2 |
20090073435 | Tsukuda | Mar 2009 | A1 |
20100045980 | Tsukuda | Feb 2010 | A1 |
Number | Date | Country | |
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20140085628 A1 | Mar 2014 | US |