KR20020016828A - Mixing apparatus and method of mixing - Google Patents

Abstract

Mixing apparatus, apparatus, and device for performing quantitative analysis. The apparatus comprises a sample chamber comprising a first and a second inlet, an inlet port for receiving a filter and / or binder holding means, and a paddle undergoing a reciprocating motion, the inlet port being in liquid communication with each inlet. Movable relative to the first and second inlets.

Description

Mixing apparatus and mixing method {MIXING APPARATUS AND METHOD OF MIXING}

Applicants have developed devices, instruments and devices for performing quantitative analysis as disclosed in PCT / GB98 / 03586. The apparatus has a first inlet, a second inlet, an inlet port, the inlet port being movable relative to the first and second inlet so as to be in selective liquid communication with the respective inlet. Also accommodates filter means and / or binder retaining means.

For example, during a quantitative analysis to determine the presence or absence of one or more analytes in a sample, the sample is separated into a first component fraction and a second component fraction, the second component fraction being It is obtained by eluting a component "held" in the binder holding means from the binder holding means.

In this application, the eluting step, in which the elutant fills the second inlet under gravity, results in a non-uniform sample (as it forms the eluting gradient in the second inlet), which causes the sample to be "read" by the measuring instrument. "It turned out to cause inaccurate readings when losing. The measuring instrument here comprises, for example, a microprocessor operable via a keypad, one or more light emitters and one or more light detectors, a display device and driver, an analog-to-digital converter and a device that connects the power source. It can be configured by means.

The present invention relates to an improved mixing apparatus and mixing method.

More particularly, the present invention relates to an improved apparatus, instrument, device and quantitative methodology for performing quantitative assays.

In a particularly preferred embodiment the invention relates to a device suitable for use in quantitative analysis of assays such as glaciated proteins in samples such as blood.

However, one of ordinary skill in the art will recognize that the basic principles of the present invention can be applied to solving mixing problems in various devices, instruments or devices.

1 is a perspective view of an apparatus according to one aspect of the present invention.

2 is a partial cutaway view of the apparatus of FIG. 1;

3 is a perspective view showing the base portion of the apparatus of FIG. 2 to show the paddle of the present invention.

4 is a perspective view of a mechanism for use with the apparatus of FIGS. 1 to 3.

One object of the present invention is to provide a simple method of making the gravity fed fraction uniform, and in particular to provide an improved machine, apparatus and / or device which can perform such a method.

According to a first aspect of the invention, there is provided a method of mixing a sample in a chamber comprising positioning a paddle in a sample and causing the paddle to reciprocate.

It is another object and independent object of the present invention to provide an apparatus, instrument or device capable of mixing a sample to be used for quantitative analysis in which an analyte is detected by spectrophotometric means and to be used to achieve this object. To provide components.

According to a further aspect of the invention, a paddle is provided which has a liquid moving surface and means for supporting the paddle in or on the chamber such that the paddle can reciprocate in the chamber.

Preferably, the means for supporting the paddle in or above the chamber consists of a pair of arms extending from the liquid transfer surface.

Preferably, the paddle is T-shaped.

More preferably, the surface for liquid movement is such that an opening is formed therein so that the light beam can pass therethrough.

In one embodiment, the paddle is made of magnetic material and is reciprocated by electromagnetic means such as a solenoid.

Of course, other mechanisms can be used to perform the reciprocating motion. For example, the paddle may be composed of piezoelectric material such that reciprocation is caused by localized current.

According to another aspect of the present invention, a sample container suitable for receiving a paddle is provided, wherein the paddle is mounted in or on the container to reciprocate in the container.

Preferably, the sample container comprises a bottom surface, side surfaces extending from the bottom surface to constitute the chamber, and means for supporting the paddle as well as configuring the side surfaces, such as a pair of slots, for example. do.

Preferably the sample container is a device comprising an optical chamber.

More preferably, the sample container is part of a carousel or cassette.

According to another aspect of the invention, there is provided an instrument suitable for receiving a sample vessel comprising a paddle, the instrument comprising means for causing the paddle to reciprocate within the vessel.

Preferably, said means for causing the paddle to reciprocate is electromagnetic means, for example solenoids.

According to another aspect of the invention, a device is provided that comprises a reading instrument comprising means for reciprocally driving a paddle in an apparatus comprising an optical chamber.

Examples of devices and mechanisms that can be adapted to suit the present invention are disclosed in PCT / GB98 / 03586.

The apparatus and apparatus described in PCT / GB98 / 03586 may be vulnerable to other problems common to the devices used for quantitative analysis. Thus, a separate problem of the general type of device described in PCT / GB98 / 03586, ie the device in which the sample or samples are provided to the instrument for reading, is that a sample, eg For example, one or more photo emitters and one or more photodetectors constituting the reading means are accurately positioned.

It is therefore an independent object of the present invention to provide a device that allows accurate readings to be made.

According to an independent aspect of the present invention, there is provided a device comprising an instrument for reading one or more samples, and an apparatus for providing one or more samples to the instrument, wherein the device is placed in a read position. This is done using two phased recognition.

Anomaly recognition preferably uses two or more independent micro switches.

The first switch informs the instrument that the device is in range, and the second switch ensures correct alignment.

The first micro switch on the instrument is operated by one "element" on the device, which constitutes the first phase of detection. Preferably, the element on the device is a protruding member that presses the micro switch mounted to the board via a rocker arm assembly. The rocker arm actuation actuates the switch even if there is any error in the horizontal position of the switch on the circuit board.

The second switch on the instrument functions as a "fine tune" which is activated when the device reaches the correct (opposite ") position on the instrument.

One embodiment of the two members constituting the switch is a resilient member to arm on the notch and mechanism on the outermost wall of the device, in particular of a carousel or cassette type device. When the carousel or cassette type device moves into position, the resilient member or arm moves from the biased position to the unbiased position and thus deactivates the switch.

This two-step recognition facilitates assembly and improves reliability of operation. It also improves the ease of use.

In the case of the carousel device of the type disclosed in PCT / GB98 / 03586, it is preferable that there are a plurality of such switches. More preferably there are four such switches at 90 ° apart.

Another problem with the general type of device disclosed in PCT / GB98 / 03586 is how to achieve good readings when measuring the amount of two different fractions.

For example, in the case of diabetes management, it is desirable to determine the percentage of frozen blood hemoglobin. This means that two quantitative results need to be obtained and a comparison between them should be made. For example, a comparison between frozen hemoglobin and unfrozen hemoglobin.

Typically the analytes were measured at the peak frequency. In the case of frozen proteins containing hemoglobin pigment, this peak frequency is on the order of 405 nm. The inventors have determined that significant advantages can be obtained by measuring off-peak, by measuring at 415-460 nm, more particularly at about 440 nm for frozen hemoglobin protein. This frequency range corresponds to the shoulder of the graph of absorbance versus wavelength for hemoglobin. 440 nm is a preferable wavelength. This increases the linear response to cover a wider and therefore more useful range of hemoglobin concentrations.

According to this independent aspect of the invention, separating a blood sample into a first component portion containing one or more non-icing proteins and a second component portion containing one or more icing proteins, more preferably at 405 nm to 460 nm Preferably, a blood glycation percentage determination method is provided which consists in detecting / quantitating an analyte by spectrophotometric means at approximately 440 nm.

The " off peak " measurement allows to avoid complicated calibration steps. For instrument performance and when comparing tests between instruments, it is important that there is a linear response between the measurement and the concentration of absorbing material. It is equally important that the range of linear responses is wide enough so that, for example, measurements of both frozen and unfrozen quantities can be made in the linear portion of the response curve. This is because the slope of the linear response varies from instrument to instrument for various reasons. Also, the slope of the linear response varies as a function of temperature or other environmental factors even within the same instrument. However, the nature of the calculation of the glyph percentage in a given instrument is that the slope of the response is canceled out. Thus, the difference in slope does not affect the results within the instruments or between the instruments unless significant changes occur during the quantitative analysis period. Thus any residual differences between instruments can be equalized using a calibrated offset made at the time of initial installation.

Using a narrow wavelength yields a linear response but reduces the response range because it approaches the absorbance maximum of hemoglobin. This is because the system is at its maximum sensitivity at this point. Selecting a suitable bandpass filter outside of the absorbance maximum extends the working response range, which reduces the sensitivity of the system and allows to remove the slope factor as described above. The reduced sensitivity is then offset by achieving a high signal to noise ratio on the detector electronics side.

Another independent problem of the general type of device disclosed in PCT / GB98 / 03586 is to ensure the accuracy of the reading and to keep the critical values (CV's) to a minimum.

The inventors have shown that the main factor influencing the threshold is to ensure that all of the samples are collected for measurement. Therefore, if a small quantity is measured, a drop of one can lose as much as 8% of the total quantity.

According to this independent aspect of the invention, one or more means for breaking the surface tension of the liquid droplets are incorporated to ensure that the liquid droplets leave the first component part of the device and enter the second component part. A device is provided.

In one embodiment the means consists of a web of webs positioned between the first and second component parts.

More preferably, the device is such that the inlet port is movable relative to the first and second inlets, the inlet port is funnel-shaped and houses the filter means or the provider holding means, the spider web being of the funnel-shaped one. It is located across the exit.

Another separate and independent problem with devices of the type described in PCT / GB98 / 03586 is that the device must remain firmly in place when reading is made.

The inventors solved this problem by carefully designing the device and the apparatus.

Thus, in one embodiment the carousel device has a tapered circumferential ring and the instrument has spring clips that pull the carousel downward to prevent wobble.

An apparatus for reading one or more samples in accordance with this independent aspect of the present invention, and an apparatus for providing the one or more samples to the instrument, the apparatus being rigidly held at a fixed position in the instrument by spring clips in the instrument. Is provided.

The main aspects of the present invention and various independent aspects of the present invention are exemplarily described below only in connection with the general type of device described in PCT / GB98 / 03586 and only in connection with the method of quantitating the amount of frozen and unfrozen hemoglobin. Will be described.

Referring to FIGS. 1 and 2, the carousel device 31 is composed of a bottom 2 (shown in detail in FIG. 3) of clear plastic, a top 6 and a funnel 32. The funnel portion 32 is made of hydrophobic plastic and has a relatively large aperture to facilitate emptying of the reagents therein. The funnel portion has an outlet 34 which directs liquid into the optical chambers 3 and 5 when the device is rotated in the instrument. The outlet 34 includes a frit (not shown) to retain particles such as, for example, an amino phenyl boronate agarose affinity matrix. The funnel 32, which functions as an inlet port, has an annular rim 36 having a recessed portion 38. The rim 36 is located partially covering the holes 40, 42, 44 formed in the upper part 6 of the device, with the tubes located vertically in the device having holes in the annular rim 38. Partially cover the holes so that they do not pass through them until they are aligned with. A bag 48 having a female mating member protrudes from the bottom of the funnel, the instrument 24 having a male member 50 adapted to engage it, so that the device 31 has a bag. It is connected to the instrument 24 via 48. The male member 50 forms a carousel in which the bottom 2 and the top 6 of the device 31 rotate around the funnel and the annular rim 36 of the funnel acts as a guide means. Keep the funnel in a fixed position for 24).

The bottom part 2 of the device consists of a transparent plastic, which is generally annular and divided into a plurality of rooms. As can be seen in FIG. 3, there are two optical chambers 3 and 5 and a third chamber 4 for receiving waste from the cleaning step, which is the optical chamber 3 and the optical chamber 5. It is located in between. And there are three additional chambers 40 ', 42', 44 'containing the reagent tubes. These chambers 40 ', 42', 44 'located below the holes 40, 42, 44 of the upper part 6 of the device 31 allow the reagent tubes to be used when the carousel is in the proper use position. Is arranged to be provided to. The optical chambers have a curved outer wall 52 and an optical quality curved inner wall 54, which collects light from the LEDs of the instrument 24 to help pass the sample in the chamber to the opposing photodiode.

Each optical chamber 3, 5 may be in liquid communication with an outlet 34 of the funnel inlet port 9. Alternatively, the optical chambers may be recessed. There is a guide member 58 extending outwardly from the outermost wall 56 of the bottom 2, which is a circumferential channel member 60 formed on the outermost wall 62 of the annular recess 64 of the instrument 24. Put on). The communication channel 66, which extends from the channel member 60 in the outermost wall 62 to the top surface 68 of the instrument 24, causes the guide member 58 to become a channel member when the device 31 is connected to the instrument 24. Allow to be inserted into (60).

The protruding members to tabs 70 on the jagged edges 72 of the upper part 6 serve as indicator means for indicating the position for positioning the device on the instrument and assist in rotating the device.

The bottom part 2 is connected with the top part and the funnel part lies in a channel formed by the steps on the top surface 78 of the top part 6.

The instrument shown in FIG. 4 is designed for use with the basic device described above. The instrument is provided with a power regulation and monitoring circuit such that the instrument can be connected to, for example, an external direct current source or a car battery. In addition, the instrument may be provided with a communication system, for example RS232, to provide a means for sending and receiving commands and for downloading data.

Means for receiving the device are annular recesses 64 in the mechanism defined by the floor, outermost wall 62 and innermost wall 80.

The floor of the annular recess includes a ramp 82 in part thereof. Within the outermost wall 62 of the annular recess is a channel member 60 and a connecting channel 66 extending therefrom.

In use, the device is inserted into the annular recess 60 by aligning the guide member 58 with the connecting channel 66 such that the device is connected to the male coupling member 50 via its arm coupling member 48. In this way, the guide member 58 can enter the channel member 60 to be rotatable. Once rotated, the first tube exits its hole 44 upwards over the inclined portion 82 because the recess 38 of the annular ring 36 is aligned with the hole. In this position the outlet 34 is in liquid communication with the first optical chamber 3 and a first step of quantitation can be performed. Rotating the device 90 ° further provides a wash solution for use through the aperture 42 and then rotating the device 90 ° further provides an eluting solution. In this way, appropriate reagents are provided for each step of the quantitation process.

The preferred apparatus and apparatus have been briefly described and now the improvements will be discussed in more detail.

Testing of the device described in PCT / GB98 / 03586 showed a threshold of 6-7% order. This was mainly due to the elution gradient formed when the frozen portion eluted from the solid phase. In fact, it was found that the frozen amount eluted at a decreasing concentration as the elution buffer was filtered and entered into the optical chamber 5. These tests indicated that the first dark droplets from the funnel 32 had gathered in the corners of the optical chamber 5 and did not mix sufficiently with the subsequent thinner droplets. As a result, the measurements made before mechanical mixing of the solution showed little "contrast" with the measurements made after mixing.

In order to overcome this problem, it has been found necessary to introduce a mixing step.

However, conventional methods have proved inadequate. For example, the device could not be shaken without fear of damaging the instrument, and such as the use of rotating ball bearings could damage the optical chamber.

The inventors have solved this problem using the paddle 100. Several approaches were used.

Having a stirring device seemed to be the main problem to be solved. Attaching the stirring component to the side walls of the optical chamber seemed to be a possible way to solve this problem. Two options were explored. In one embodiment, the paddle is fixed on the sidewall of the optical chamber and made to vibrate in the optical axis direction using an electromagnet. A hole in the center of the paddle provides a passage for light from the LED.

In another embodiment the paddle is fixed on one side of the optical chamber 5 and made to oscillate perpendicular to the optical axis away from the passage of light.

All of these embodiments adjusted the frequency of the oscillator driving the electromagnet to provide adequate mixing once the resonant frequency was found. Although attractive, this method still had some problems.

Paddles must have stiffness, so a significant amount of energy is required to generate vibrations. This may be related to any battery operated apparatus.

Furthermore, the resonant frequency varies from component to component and depends on the liquid level in the chamber. Thus, some means of scrutinizing the resonant frequency will be required to ensure proper mixing by matching the resonant frequency. Both components also had a three-dimensional shape, which required more cost.

An alternative to using flat paddles has overcome the problems associated with the vibrating method described above.

Thus, in the preferred embodiment, the metal paddle 100 is retained in the grooves 101 formed as the side walls 102 and 104 of the optical chamber 5 are erected as shown in FIG. 3. The paddle was able to reciprocate with minimal friction and was forced to swing through the solution along the optical axis direction using an electromagnet located below the photodiode on the outer circumference of the platen molding, which will be described later. Could be added. Holes 106 are provided to enable light from the LED to reach the detector.

Moving the paddle requires a very small force, so significantly less energy is required to drive the electromagnet. Experimental results showed that up to 10 swings were required to make the layered dye and water starting solution into a uniformly visible solution.

The lower surface of the upper molding 6 has been shown to effectively hold the paddle by placing a spider web (not shown) just above the center of the paddle.

Another improvement involves the use of two micro switches in a phased approach. This allows the device 31 to be detected accurately and without ambiguity in the instrument 24. The switches (not shown) at each of the four locations are actuated by a plastic cylindrical columnar feature 58 (in this case also a guide member). This pushes the microswitch (not shown) installed on the board through the rocker arm assemblies 110, 112, 114 and 116 (FIG. 4) in each of the four operating positions. The rocker arm operation overcomes any error in the horizontal position of the switch on the circuit board (not shown).

The second phase of detection is provided by a microswitch actuated by the action of the not shown ratchet arm and the respective notches or notches 120 and 122 (only two of the four shown) on the outermost wall 56 of the carousel. do. A flange 131 extending from the ratchet arm contacts one switch on the instrument. The ratchet arm is broken by the outermost wall 56 of the carousel when the carousel is in one of the four operating positions and the ratchet arm enters the notch of the carousel to deactivate (disable) the switch, but not in these positions. The force is applied to make the switch work. These notches are preferably shaped to allow rotation in only one direction. These switches are only deactivated when the instrument and device are in the correct place. This two phased approach facilitates assembly, improves reliability of operation and increases ease of use.

Finally, another improvement relates to the structure used to overcome the "wobble" problem. Any movement between the carousel and the instrument, however small, may change the path of the light during reading. This reading problem could be overcome by modifying the carousel and the instrument to provide a locking arrangement.

In one embodiment, the carousel has a cylindrical ring 124 composed of an inclined surface 126 and a flat surface 128.

The mechanism 24 for receiving the carousel has a casing 130 and a printed circuit board 132, which is mounted on the printed circuit board with a platen 134 and presses down consisting of four spring clips 138, 140, 142 and 144. A hold down 136 is installed. When the carousel is inserted into the instrument, the spring clips climb on the inclined surface 126 to press and secure the flat surface 128 with its claws.

Claims (35)

Positioning the paddle (100) in the sample and causing the paddle to reciprocate. The method of claim 1, wherein the paddle is made of a magnetic material and comprises means for supporting the liquid moving surface and the paddle in or on the chamber, the paddle being made to reciprocate by the action of an electromagnet means. . 3. The device of claim 2, wherein the means for supporting the paddle in or on the chamber comprises a pair of arms extending from the liquid moving surface, wherein the arm has a pair of slots on the side extending from the bottom defining the chamber. The sample mixing method, which rests upon 4. The method of claim 2 or 3, further comprising passing a light beam from the light emitter through an opening 106 formed in the liquid moving surface of the chamber and the paddle to a photodetector to detect the analyte in the sample. Sample mixing method. The method of claim 4, wherein the sample is frozen hemoglobin and is detected by spectrophotometer means between 405 nm and 460 nm. Paddle (100) comprising a liquid moving surface and means for supporting in or above the chamber to reciprocate in the chamber. 7. The paddle of claim 6, wherein the means for supporting the paddle in or on the chamber consists of a pair of arms extending from the liquid moving surface. The paddle according to claim 6 or 7, wherein the paddle is T-shaped. The paddle according to any of claims 6 to 8, wherein the liquid moving surface has an opening (106) for the light beam to pass through. 10. The paddle according to any of claims 6 to 9, wherein said paddle is made of magnetic material. 10. The paddle according to any of claims 6 to 9, wherein said paddle is made of piezoelectric material. And a chamber (3,5) made to receive a paddle (100), wherein the paddle is mounted in or on the chamber to allow the paddle to reciprocate in the chamber. 13. The sample container of claim 12, comprising a bottom defining a chamber and a side extending therefrom, the sides comprising means for supporting a paddle. The sample container of claim 13, wherein the means for supporting the paddle is a pair of slots on the sides. 15. The sample container of any of claims 12-14, wherein the chamber is an optical chamber. 16. The sample container of any one of claims 12-15, wherein the sample container is a carousel or cassette. 17. The carousel according to claim 16, wherein the sample is a carousel for use in quantitative analysis provided in the instrument by separating the first component and the second component, the first inlet in or guided in the first component volume collection chamber, And a second inlet in or guided by a second component collection chamber and an inlet port for receiving filter means or binder holding means, said inlet port alternating with each of said first and second inlets as desired. A sample container movable relative to each of the first and second inlets so as to be in liquid communication therewith. 17. The carousel according to claim 16, comprising a bottom having a plurality of chambers comprising a first and a second inlet, an upper part constituting the carousel with the bottom, and a funnel portion comprising an inlet port, A sample container, rotatably mounted relative to the funnel. And a means adapted to receive a sample container comprising a chamber adapted to receive a paddle, the apparatus further comprising means for causing the paddle to reciprocate within the chamber. 20. The instrument of claim 19 wherein the means for causing the paddle to reciprocate is electromagnetic means. 21. The instrument of claim 20 wherein the electromagnetic means is a solenoid. And an instrument capable of detecting an analyte in a sample provided in a sample container comprising a chamber adapted to receive a paddle, wherein the paddle is initiated by the instrument to allow for reciprocating motion or in the chamber. Mounted on, device. An instrument for reading one or more samples and an apparatus for providing the one or more samples to the instrument, wherein positioning the one or more samples into a reading position is accomplished using anomaly recognition, device. The device of claim 23, wherein the first switch informs the instrument that the device is in range and the second switch verifies correct alignment. 25. A device according to claim 23 or 24, wherein the first micro switch on the instrument is actuated by some "element" on the apparatus, which constitutes the first phase of detection, and the second switch on the instrument functions as "precision adjustment". Wherein the device is activated when the correct position on the instrument is reached. 27. The device of claim 25, wherein any of the " elements " on the device is a protruding member that presses a micro switch mounted to the board through the rocker arm assembly. 27. The device of claim 26, wherein the two members of the switch are notches in the outermost wall of the device and elastic members to arms on the mechanism. 28. The device of claim 27, wherein the device is a carousel to cassette type device. The device of claim 28, comprising four switches spaced 90 degrees apart. The blood sample is separated into a first component portion comprising one or more non-freezing proteins and a second component portion comprising a frozen protein of the one or more proteins, and detecting / quantifying frozen hemoglobin by spectrophotometer means from 405 nm to 460 nm. A method of determining the percentage of glycation in the blood, including. 31. The method of claim 30, wherein detecting / quantifying frozen hemoglobin is measured at approximately 440 nm. An apparatus incorporating one or more means for breaking the surface tension of the droplets to ensure that the droplets exit the first component portion and enter the second component portion. 33. The apparatus according to claim 32, wherein said means consists of webs shaped like positioned between the first and second component parts. 34. The device of claim 32 or 33, wherein the device is movable relative to each of the first and second inlets in which the inlet port is in or directs to the first and second collection chambers, the inlet port being funnel And a filter means or a binder holding means, wherein the cobweb is located across the outlet of the funnel. A device comprising an instrument for reading one or more samples and an apparatus for providing one or more of the samples to the instrument, the device being held firmly in place within the instrument by spring clips.