AU742119B2 - Spectrophotometry apparatus - Google Patents

Description

P/0 0/0 11 Regulation 3.2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT 0.00 5 0 00 0 SS 00 SPECTROPHOTOMETRY APPARATUS VARIAN AUSTRALIA PTY LTD

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000555

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5.6050 0 55 S S 055 5 The following statement is a full description of this invention, including the best method of performing it known to me: JM CAkWINWORDUENNYM\JOHN\!R49S 2 SPECTROPHOTOMETRY APPARATUS This invention relates to electric circuitry for enabling very accurate measurement of temperatures of samples such as may be contained in a carrier for use in an instrument for spectrophotometrically analysing the samples. The invention is described herein with reference to an exemplary application, namely in analysing DNA, however this exemplary application is not to be regarded as limiting on the invention as many other applications for the invention are possible.

Throughout this description, the word "comprise" and variations of the word such as "comprises" and "comprising", is not intended to exclude other additives, features, components or integers.

One way of characterising DNA is to measurevery accurately the temperatures at which optical density changes occur in the DNA. Such optical 15 density changes can be detected by measuring the absorbance of samples :o using a spectrophotometer. Thus spectrophotometric typing of DNA may •characterise double strand DNA (dsDNA) with a defined length by determining the denaturation or renaturation temperatures and length of melting-domains.

Generally, the optical density of DNA increases as denaturation occurs, 20 providing discrete, quantifiable changes in absorbance. As renaturation occurs, optical density decreases.

In characterising DNA by measuring optical density changes at varying ooooo3 temperatures, a very high resolution for the temperature measurements is required. This resolution may need to be accurate to 1/100 of a degree.

Achieving this level of temperature accuracy is very difficult.

Spectrophotometric analysis of DNA offers the possibility of fast analysis and hence increased productivity over other analysis methods. This is because it allows many samples to be analysed in parallel in one run rather than every sample requiring separate processing. For a batch of, for example 48 samples to be measured over a 20 0 C temperature range to 1/100 of a degree resolution will involve 96,000 optical density measurements. For these measurements to be made within, for example, two hours will require at least 13 measurements ,per second to be made. For this measurement rate and because control of V Le1erpiatue Lo the required accuracy is slow, measurements will need to be

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o° oooo oo oo ooooo o oe oo o oooe oooe ooo o ooo oooo oooo 3 made on all samples at the same temperature before moving to the next temperature. This means that 13 measurements per second will be made on 48 different samples at the same temperature. Thus the 1/100 of a degree temperature resolution has to be established within a sample and between all 48 samples. This level of temperature accuracy and uniformity for such a typical number of samples at such a measurement rate is very difficult to achieve.

The provision of a sample of DNA for spectrophotometric analysis may include or require an initial step of amplifying the DNA by the polymerase chain reaction (PCR). This reaction requires accurate temperature control combined with rapid heating and cooling in that the starting DNA together with selected additives is cycled between three temperatures according to a specific timetemperature profile. Conduct of a PCR within a cell that is also used to measure the optical density of the DNA at varying temperatures is desirable as it avoids the need to transfer samples between cells.

The high level of precision in temperature control required for amplifying and/or analysing DNA samples requires that temperatures of the sample carrier and possibly a housing for containing the sample carrier be able to be measured to the same level of precision. Conventional methods of measuring 20 temperature using platinum resistance thermometers in a bridge configuration and compensatory electronics circuits to eliminate known non-linearities of the thermometers, cannot provide the required level of precision. This is because there are too many individual component tolerances all of which increase the error. At best 1/10 of a degree accuracy is possible with a conventional approach whereas the preferred field of use for the present invention requires 1/100 of a degree accuracy.

From the above, there is clearly a need for accurate measurement of temperature and the object of the present invention is to provide a temperature measurement circuit to meet that need.

According to the invention there is provided a temperature measurement circuit for use with a sample carrier or a housing for containing the sample carrier, wherein the circuit comprises a resistive temperature sensing element for measuring the temperature of a region (eg. the carrier or the housing), a first S precision fixed value resistor, a second precision fixed value resistor connected

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DoCUTien=lt2 0 Ollltn2 4 in series with a current source, and common circuitry for measuring voltage drops across the temperature sensing element and the first and second resistors for ratiometrically calculating the temperature of the region from the measured voltage across the temperature sensing element and the known values of and measured voltages across the first and second resistors.

Preferably the temperature measurement circuit includes first and second resistive temperature sensing elements and the first and second precision fixed value resistors all in series with the current source for measuring, respectively, the temperature of a first region (eg. the sample carrier) and a second region (eg. the housing). The temperature of the second region can be ratiometrically calculated from the measured voltage across the second temperature sensing element and the known values of and measured voltages across the first and second resistors. The resistance-temperature characteristic of the resistive temperature sensing elements will, of course, be 15 known and can be allowed for in a computer software algorithm for performing the required calculations.

•A key issue in this invention is the use of common circuitry in measuring the signals from these resistive components. Preferably this common circuitry comprises a switching network for referring voltage levels to a differential ooooe S 20 amplifier wherein the differential amplifier output signal represents the voltage Co across a temperature sensing element, the first or the second resistor, as determined by the switching network.

Most of the sources of error contributed by componentry in prior art arrangements are eliminated in the above described circuit by virtue of the fact that such errors are made common to all measurements and are therefore cancelled out.

Preferably the resistance value of the first precision fixed value resistor is chosen to correspond substantially with the resistance of the temperature sensing element(s) at a temperature substantially equal to the lower temperature limit of the expected temperature measurement range and that of the second precision fixed value resistor to correspond substantially with the resistance of the temperature sensing element(s) at a temperature substantially equal to the upper temperature limit of the expected temperature measurement uL}S ra n g e C Preferably the output of the differential amplifier of the common circuitry is referred to an analogue to digital converter and is then fed into a computer for performing the ratiometric calculations. Preferably the algorithm for performing the calculations eliminates known non-linearities of the resistive temperature sensing elements. Preferably the resistive temperature sensing elements are platinum resistance thermometers.

A sample carrier with which the invention may be used will typically include a number of cells, each for containing a sample for analysis.

The sample carrier with which the invention is useable may include a temperature sensing element and preferably this is provided at or in one of the sample cells to measure the temperature at an actual sample position, thereby ensuring as accurate a measurement of sample temperature as possible.

Preferably this temperature sensing element is a platinum resistance thermometer element which is placed in a cell and bonded to the body of the 15 carrier. It may be a component which is part of the electronic temperature S•measurement circuit of the invention.

eeoe° A housing with which the invention is useable is for controlling the temperature of the immediately surrounding environment of a sample carrier for achieving very accurately controllable heating regimes for samples in the cells S 20 of the carrier. A housing, within which the carrier is positionable, may include heating/cooling means for controlling its internal temperature. Preferably this housing is a separate component which, along with a sample carrier positioned °°eoo °therein, is itself positionable in spectrophotometric analysis apparatus.

Alternatively it may be a fixture of the spectrophotometric analysis apparatus.

Ideally the housing is constructed of a material possessing good thermal conductivity having a sufficient cross-section, at least for its base, to maximise the uniformity of its heat distribution over the entire housing, for example, the housing may be made of aluminium and have a base which is for example, between 4 to 15mm thick. The housing may include, or its immediate surround may comprise, outer insulation material for thermally insulating the housing from the external ambient environment. Inner surfaces of the housing may also be lined with insulating material to isolate thermally the carrier from the housing structure as much as possible.

btcw, 6 The heating/cooling means of the housing for controlling its internal temperature may be at least one Peltier heating/cooling element attached to the housing or a plurality of such elements attached to the base of the housing.

The housing may also include a temperature sensing element for measuring the internal temperature of the housing, which is a component of the temperature measurement circuit of the invention.

The above description will be clarified by the following illustrative description of embodiments. First, an embodiment of a sample carrier will be briefly described with reference to Fig. 1. An embodiment of a housing will then be briefly described with reference to Fig. 2 and an embodiment of a temperature measuring circuit according to the invention will then be described with reference to Fig. 3.

Fig. 1 shows a carrier 10 comprising an annular body 12 having an upper surface 14, a lower surface 16 and a cylindrical peripheral outer surface 18 joining surfaces 14 and 16. Body 12 is mountable in a spectrophotometer for rotation about a central axis 13 which, in use of the carrier, is substantially vertical. For this purpose it may include a lip 15 extending inwardly from the inner wall 19 of the carrier at upper surface 14 allowing for the attachment of a support and hub structure (not shown). A plurality of holes 20 extend into body 12 from upper surface 14. Holes 20 are equally spaced both circumferentially and radially from the central axis 13 to thereby define a circle formation adjacent peripheral surface 18. The holes 20 define sample cells and may extend part way through body 12 in which case the body 12 must be transparent to allow for the passage of light through each cell 20 for making absorbance measurements in a spectrophotometer. Alternatively, the holes may extend all the way through the body 12, in which case the body will have a base 22 attached to its lower surface 16 which provides a bottom for each cell If body 12 is formed of an opaque material, eg. titanium, then base 22 must be transparent, eg. glass or quartz, to allow for the passage of a beam of light through each cell from the bottom to the top (or top to bottom) of each cell The cylindrical peripheral surface 18 of body 12 includes outwardly directed rims 24 at its upper and lower edges respectively for accommodating W: DELILAH\DCNODELE\91363-98a.doc ~FFIC~ -the turns of a heating coil wound onto the body. The facing surfaces of rims 24 7 respectively, (ie. the rims have a narrow thickness) to ensure the heating winding covers as much as possible of the area of peripheral surface 18.

One of the cells 20 includes a resistive temperature sensing element 26 bonded to the wall of the cell and thus to body 12.

In normal use, the energy input to a carrier from the measuring light passed through each cell is negligible and has no effect on the temperature control regime.

A housing 28 as illustrated in Fig. 2 comprises a base 30 (eg. of relatively thick aluminium as described hereinabove) and a cylindrical side wall 32. Base 30 includes an aperture 31 for passage of the hub of a carrier 10 allowing for fast rotation of the carrier within the housing. Attached to the base 30 are Peltier heating/cooling elements 34. The inner surfaces of base 30 and side wall 32 may be lined with an insulating material. External insulation, for insulating the housing from the ambient environment, may be provided on wall 15 32 or by a further enclosure.

Two series of fins 36, which are oppositely located, extend outwardly of side wall 32. The fins 36, which are located on the Peltier elements 34, define channels 38 that extend from the outside to the inside of the housing 28. These fins 36 provide the above described portions of the housing that are left 20 uninsulated to allow thermal contact between the housing and a gas inside the housing. A fan (not shown) can be used to direct a gas over the fins 36 into the housing via channels 38. This gas can enter via one series of channels 38 and exit via the oppositely located series of channels 38. The housing includes an aperture 40 for accommodating a resistive temperature sensing element (this aperture 40 is illustrated as being in base 30, but could be through side wall 32 or elsewhere located to measure the temperature of a gas in the housing).

The base 30 of housing 28 also includes at least one aperture 42 for passage of light to be used for the spectrophotometric analysis of samples within cells 20 of a carrier A temperature measurement circuit 44 as shown in Fig. 3 comprises a stable current source 46, a first resistive temperature sensing element 48, first and second precision fixed resistors 50, 52 and a second resistive temperature sensing element 54 connected in series, as shown. The first and second i u/US resistive temperature seinsing elements 48 and 54 may be those assoUiaLU l :\DELILAH\DCNODELE\91363-98a.doc OVc W Ft- 8 with the carrier 10 and housing 28. Two solid state switches 56, each of four poles, are ganged and connected to the circuit 44 components 46 to 52 as shown by connections 57 to refer source voltage levels to the inputs of a differential amplifier 58. Switches 56 are operable by two digital inputs 59 to select one of four source voltages each such that an output from amplifier 58 represents the voltage across one or more of the components 48 to 54, depending on which sources are selected by the switching networks 56. In Fig.

3, the connections between components 48 to 54 and switches 56 are such that the measurements are across R 1 and then (R1 R 2 instead of R 1 then R 2 The illustrated arrangement is preferred as it yields substantially better accuracy than an arrangement which measures voltages across R 1 and then R 2 The output of differential amplifier 58 is then suitably amplified via amplifier 60 and converted to a digital reading by A/D converter 62 and referred to a computer S° 64 in which the resistance of each sensing element may be computed by 15 interpolation from the measured voltages corresponding to each resistance.

The temperature measured by the resistive temperature sensors 48 or 54 is then determinable from the computed resistance for each sensor.

In the circuit of Fig. 3, current source accuracy, A/D converter offset, A/D and amplifier gain scaling, are all eliminated by making them common to all S 20 readings and therefore cancelled out. The only components left are the two resistive temperature sensing elements 48 and 54, the two precision resistors 50 and 52 and the A/D converter linearity. Compensation for non-linearity of the resistive temperature sensing elements 48 and 54 can be readily compensated 2 exactly via a software algorithm in computer 64 implementing a manufacturer's non-linearity compensation formula.

A preferred implementation for means on a carrier for standardising the output of a platinum resistance thermometer (PRT) also included on the carrier, is represented by the circuit shown in Fig. 4. If a is defined as

R

100 a Ro where R 100 is the resistance of the PRT on a carrier at 1000C, and Ro the puSj resistance of that PRT at 00, and k is defined as a fixed target for a (given a b varies from one PRT to another), it can be shown that W:\DELILAH\DCNODELE\91363-98a.doc O~ffl 9 a (k-1)R o 3 a-k (where R 3 R1 R 2 Hence R 3 can be adjusted so as to keep k constant for PRT's of varying aX.

It can also be shown that'for a current i at 0°C Vo,, RoR 2 i Ro

R

3 If Vout at 0°C is set to be the same as the voltage developed across an ideal PRT of resistance C ohms, then it can be shown that C(R+ R 3

R

2 Thus R, R 2 is chosen to adjust k, and then R 2 chosen to compensate for variations in Ro.

In practice R, and R 2 need to be 0.1% resistors to achieve the required 15 level of compensation. Also the target k must be less than the lowest a and the target C must be less than the lowest Ro for R 1 and R 2 to have realizable values (ie. greater than 0 ohms). Electrical connection to the circuit (which, being on the carrier, will rotate therewith) can be made via known low noise means for connecting between stationary and moving parts of a circuit.

20 A further aspect of the invention relates to the association of a compensating means and a temperature sensing element (for example as illustrated by Fig. 4) to provide an entity in its own right, that is, to provide a means for standardizing the output of a resistive temperature sensing element, along with the element, independently of them being mounted on a carrier.

The various aspects of the invention described herein are susceptible to variations, modifications and/or additions other than those specifically described and it is to be understood that the invention includes all such variations, modifications and/or additions which fall within the spirit and scope of the above description.

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Claims (8)

1. A temperature measurement circuit for use with a sample carrier or a housing for containing the sample carrier, wherein the circuit comprises a resistive temperature sensing element for measuring the temperature of a region, a first precision fixed value resistor, a second precision fixed value resistor connected in series with a current source, and common circuitry for measuring voltage drops across the temperature sensing element and the first and second resistors for ratiometrically calculating the temperature of the region from the measured voltage across the temperature sensing element and the known values of and measured voltages across the first and second resistors.

2. A temperature measurement circuit as claimed in claim 1 including first and second resistive temperature sensing elements and the first and second 15 precision fixed value resistors all in series with the current source for measuring, respectively, the temperature of a first region and a second region.

3. A temperature measurement circuit as claimed in claim 1 or claim 2 wherein the common circuitry comprises a switching network for referring 20 voltage levels to a differential amplifier, wherein the differential amplifier output signal represents the voltage across a temperature sensing element, the first or the second resistor, as determined by the switching network.

4. A temperature measurement circuit as claimed in any one of claims 1 to 25 3 wherein the resistance value of the first precision fixed value resistor corresponds substantially with the resistance of the temperature sensing element at a temperature substantially equal to the lower temperature limit of the expected temperature measurement range and that of the second precision fixed value resistor corresponds substantially with the resistance of the temperature sensing element at a temperature substantially equal to the upper temperature limit of the expected temperature measurement range.

A temperature measurement circuit as claimed in any one of claims 2 to ,'herin the onf the rdfferentia! amnlifir nf th cormmon circuitn i W \D 1ILAH\DCNODELE\91363-98a.doc Y C 11 referred to an analogue to digital converter and is then fed into a computer for performing the ratiometric calculations.

6. A temperature measurement circuit as claimed in claim 5 wherein the computer includes an algorithm for performing the calculations which eliminates non-linearities of the resistive temperature sensing elements.

7. A temperature measurement circuit as claimed in any one of the preceding claims wherein the resistive temperature sensing elements are platinum resistance thermometers.

8. A temperature measurement circuit substantially as hereinbefore described with reference to Fig. 3. 15 DATED: 4 October, 2001 PHILLIPS ORMONDE FITZPATRICK Attorneys for: VARIAN AUSTRALIA PTY LTD 9 *u W L O