Apparatus for automated analysis

Abstract

An apparatus for pretreatment of a desired sample in a discrete fluid analyzing instrument includes a frame rotatably mounted on or within the discrete fluid analyzing instrument with a column for immobilizing at least one substance or analyte from the sample. The substance or analyte is reversibly immobilized. A first fluid transport line is provided for aspirating the sample into the column and for ejecting the substance or analyte eluted from the column. A pump is provided for pumping the sample through the column with a second fluid transport line connecting the pump to the column. A member is providing to supply the eluted substance or analyte to the discrete fluid analyzing instrument for measurement of at least one property of the substance or analyte. A device is provided for simultaneously or consecutively directing any non-pretreated sample or any subsample directly to the discrete fluid analyzing instrument.

Claims

1-10. (canceled)

11. An apparatus comprising: a pretreatment unit; and a discrete fluid analyzer in fluid communication with the pretreatment unit; wherein the pretreatment unit comprises a column comprising a material which retains at least one matrix substance or at least one analyte on the column from a sample introduced to the column and generates a pretreated sample therefrom; wherein the discrete fluid analyzer is configured to measure at least one property of a non-pretreated sample which has not been subjected to pre-treatment by the pretreatment unit; and wherein the pretreatment unit is configured to operate independently of the discrete fluid analyzer such that the pretreatment unit is capable of generating the pretreated sample simultaneously with the discrete fluid analyzer measuring the at least one property of the non-pretreated sample.

12. The apparatus of claim 11, wherein the pretreatment unit and discrete fluid analyzer are mounted on a frame and are in fluid communication with one another, and wherein the pretreatment unit is rotatably mounted on the frame.

13. The apparatus of claim 11, wherein the pretreatment unit comprises: a first fluid transport line through which a sample is introduced into the column; a second transport line through which the pretreated sample travels from the column; a pump for pumping the sample through the column, and the first and second fluid transport lines; and a dispenser configured to direct the pretreated sample to the discrete fluid analyzer for measurement of the at least one property of the pretreated sample.

14. The apparatus of claim 11, wherein the apparatus further comprises a sample wheel comprising a plurality of vessels comprising pretreated samples and untreated samples.

15. The apparatus of claim 14, wherein the sample wheel further comprises a plurality of vessels comprising an elution fluid for eluting a retained analyte from the column.

16. The apparatus of claim 14, further comprising: a plurality of cuvettes; a cuvette dispenser configured to transfer one or more of the plurality of cuvettes to the discrete fluid analyzer; a plurality of vessels housing one or more reagents; and a reagent dispenser configured to transfer the one or more reagents into respective ones of the one or more cuvettes.

17. The apparatus of claim 16, further comprising a dispenser for transferring one or more of the pretreated or untreated samples from the sample wheel to the plurality of cuvettes.

18. The apparatus of claim 11, further comprising a washing station for introducing a wash fluid through the column following pretreatment of a sample in the column.

19. The apparatus of claim 11, wherein the at least one property of the non-pretreated sample is measured photometrically or electrochemically.

20. The apparatus of claim 11, wherein the material of the column comprises a material which reversibly retains the at least one analyte on the column.

21. The apparatus of claim 11, wherein the pretreated sample and non-pretreated sample are subsamples of the same sample.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0062] FIG. 1 shows diagrammatically an automated discrete fluid analyzing device to which the invention is implemented.

[0063] FIG. 2 shows diagrammatically one embodiment of a pretreatment unit for device in FIG. 1.

[0064] FIG. 3 shows a flow diagram of one operation workflow of the invention.

[0065] FIG. 4 shows an explanatory screen display of an apparatus utilizing the invention.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

[0066] In the subsequent text the following terms and devices should be understood to be defined as below:

Pretreatment

[0067] Separate step from analysis chemistry; provides eg. a concentrated, fractionated or purified analyte. In general pretreatment may be modifying or non-modifying in relation to the substance to be pretreated.

Discrete Fluid Analyzer

[0068] Discrete fluid analyzer performs chemical reactions and photometric measurements in single/separate wells. Usually an analyzer contains multiple wells in which different reactions are ongoing simultaneously. Several different tests may be requested from each sample. Also different types of samples may be analyzed simultaneously.

Column

[0069] Column is typically a cylindrical or other formed vessel to which liquid samples and reagents may be introduced and analytes and/or other components eluted. The column may be in-line connected to a pump or syringe in which case the samples and reagents are pumped to and from the column using positive or negative pressure. Or the column may be freely diffusing in which case samples and reagents are drawn to and from the column by gravity or by centrifugal force. The column may be packed or lined with material that has affinity towards the analytes or matrix components. As an alternative, the column may be packed with material that functions as a sieve or filter. Physical treatments may also be connected to the column.

[0070] The column may be single use (disposable) or reusable.

Sample

[0071] A whole sample as collected is a sample or primary sample. The part of the sample that is pretreated, is a sub sample.

[0072] The substances that are analyzed are analytes. All other components in a sample are matrix.

[0073] The analytes may include, for example: ions, molecules, groups of chemically related substances, substance, family of substances such as proteins and chemical species.

[0074] Matrix includes all matter that is not measured, for example: cell debris, substances interfering with analyte measurement, analyte complex forming/binding substances that interfere with analyte measurement.

Washable

[0075] Washable means herein a device that can be successively used by cleaning it between consecutive operative steps, opposite to a single use device that is discarded after every contact with analyte or ather liquid in order to avoid contamination.

[0076] FIG. 1 shows a typical discrete analyzer. As these apparatuses have been in laboratory use and their operation principles are known to persons skilled in the art or can be easily studied from operating manuals and basic literature, only a cursory description of the apparatus shown in FIG. 1 is provided herein as an example. The invention is not limited to any specific type of an automated discrete analyzing device as long as its operation is controlled by sample handling rules permitting sample handling routines for each sample so that a request to analyze a sample may include an assay of the measurements required and a request for pretreatment if such is desired.

[0077] FIG. 1 shows the main elements of an analyzing device. These are arranged on a suitable frame and include diluent water container 1 and waste water container 2. Samples to be analyzed are placed on sample wheel 4 and reagents, enzymes and other media needed for analyses on reagent wheel 3. Samples are handled with sample dispenser 5 and reagents with reagent dispenser 6. Measurements of analytes or components within the samples occur in incubator 7 with a photometer 8.

[0078] For the pretreatment of a sample, a pretreatment unit 9 is arranged in relation to sample wheel 4. The sample wheel 4 is divided in sections that form racks 10, 11, 12 arranged as reagent rack 10 for elution and wash liquids, microplate rack 11 for pretreated samples and sample racks 12 for untreated samples. Between the sample and reagent wheels is a cuvette dispenser 13. FIG. 1 represents an exemplary arrangement with other arrangements possible.

[0079] In further description of FIG. 1, when pretreatment is not needed for the indicated measurement, cuvettes are fed from the cuvette dispenser 13 to the incubator 7 whereafter sample volumes of the collected sample are aspirated with the sample dispenser 5 into the cuvettes as requested by analyzing assay request of the sample. Accordingly, reagents needed for requested measurements are dispensed by reagent dispenser 6 into the cuvettes and after a measurement specific incubation period, the measurement is performed for each cuvette by photometer.

[0080] When the analyzing assay request includes pretreatment, a portion of the sample is aspirated in the pretreatment unit 9 shown in FIG. 1. A side view of pretreatment unit 9 is shown in FIG. 2.

[0081] The exemplary pretreatment unit of this embodiment is built on a frame 14 that is rotatably mounted on the analyzing device. The main parts of the pretreatment unit 9 may include a pump 21 for sample aspiration and ejection through line 17. Line 17 connects the pump line to a pretreatment column 16. In order to enable use of a long column 16 and sufficient pretreatment time and to keep the dimensions of the pretreatment unit 9 small, the column 16 shown in this embodiment is on a grooved stationary wheel 18. On the opposite end of the line 17, the column 16 is connected to a suction or pipetting tube 15 for aspiration and ejection of the sample.

[0082] One type of pretreatment column 16 is a column containing internal surfaces that immobilize the analyte from the sample to the surfaces of the column. These surfaces may be walls of the column itself or some filler material, which may be in particle or fibrous form. For pretreatment in such a column the substance must be first retained and then released from the surfaces and placed into cuvettes in for measurements. For this the sample wheel has a reagent rack 10 for elution and wash liquids. Further, FIG. 1 shows a washing station 19 arranged in vicinity of pretreatment unit 9. When a pretreatment is needed, the cycle may start with a washing cycle of the column 16 at the washing station 19, but usually the column 16 is washed at the end of the pretreatment cycle. Washing is performed simply by aspirating water or other washing liquid into the column so that it is substantially filled and then ejecting the washing solution into a waste container 2. The aspiration and ejection is repeated as many time as needed to obtain desired level of purity and the washing liquid may be pumped reciprocatingly in order to increase removing effect.

[0083] For pretreatment a desired amount of sample liquid is aspirated into the column 16 through pipetting line 15 and the sample is pumped through the column so that at least the entire sample volume has entered the column. This forms a sub-sample of the original sample, which remains untreated and may be used for other measurements without the effects of pretreatment. In the column, the substance, for example a molecule, is retained and immobilized on the wall or other interior surface of the column. For this, the sample may be kept in the column or pumped reciprocatingly in the column to ensure sufficient reaction time. Heating or cooling of the sub-sample may be used for enhanced effect. After the reaction time needed to immobilize the target analyte has passed, the sample is ejected from the column. The target analyte remains immobilized within the column. In order to make the measurement, the immobilized molecule or substance must be eluted from the surface via washing the column with an elution liquid placed in the reagent rack 10. The elution liquid is ejected together with the substance eluted from the column 16 to a microplate rack 11 to await transfer to the incubator or directly to the cuvette in the incubator. The measurement of the sample occurs in a routine manner, for example, by a photometer according to operating principles of the analyzing apparatus. Thus, the pretreatment step is merely one additional handling step in the analyzer for the sample and can be arranged within the normal measuring cycle without disturbing the operation of the apparatus or changing the function, construction or operation of the apparatus. The pretreatment unit and washing stations, reagents and elution liquids may be fitted to existing apparatuses without difficulty.

[0084] The main problem with handling samples for discrete analyzers is the small volume of the sample. Since the volume of the sample is small, many traditional pretreatment methods like evaporation or treatments based on chemical reactions performed on the substance to be measured are difficult to implement. The present invention provides a solution to this problem by using capture or immobilization methods for either enriching the substance that is measured or its proportion in the sample. This is accomplished by retaining the target analyte or retaining other substances that interfere with the measurement of the target analyte and removing them from the sample. The retaining method itself may vary, but the immobilization method discussed above provides one embodiment suitable for implementation for many various measurements.

[0085] Another approach to retaining a substance is adsorption or physical bonding of the substance to a surface or another substance via chemical binding. In practice binding may occur through covalent bonds, electrostatic interactions or van der Waals forces or other surface forces between molecules or substances.

[0086] Various pretreatment methods will be discussed below in further detail.

[0087] This method of using a pretreatment unit enables transfer of the treated sample into the measurement unit. Transfer is controlled by a user friendly software whereby the instrument user may simply add the primary sample tube in the instrument sample feeding unit and program the desired assay. When a sample pretreatment step is necessary, the software automatically directs the sample into the pretreatment unit, while other analyses from the same sample or other samples continue to be performed without delay or disturbance.

[0088] Within the clinical and non-clinical fields, the present invention benefits numerous assays.

[0089] For example, many physiologically important compounds including without limitation, Vitamin A, D and E, are fat-soluble and/or bound in circulation to a corresponding binding protein or other protein structures. Serum vitamin D samples must be pretreated in order to disrupt the protein complex and extract the analyte in an organic solvent such as acetonitrile or methanol by a manual procedure. With a pretreatment unit containing a surface for immobilizing the formed complex, the analyte can be eluted by a suitable solvent and then transferred to the assay performing unit. Immobilisation may be accomplished by antibody binding or by chemical binding and the surface will be automatically regenerated and washed between samples. A similar separation step may be adapted to many small molecular compounds and thus avoid using instrumentation more complicated than a photometer.

[0090] The pretreatment surface may comprise a coated surface inside a glass or metallic column, enabling solid phase extraction. Particles of uniform or different size may be coated and packed in a tube or other column container. Coated fibers or other materials may also be used. The reactive surface may also comprise a microfluistic platform or otherwise comprise liquid pathways molded or printed on various suitable materials. The coating may include antibodies or other specific binders. Also, many chemically reactive surfaces (hydrophilic, hydrophobic, lipophilic, nitrogenous or organic group binding) may be useful. In one embodiment, binding is irreversible or accomplished via bonds from which the substance can be removed and the surface regenerated or comprises equilibrium binding.

[0091] Sophisticated and time consuming chromatographic techniques are often used to measure components in mixtures, if there are no simple ways to separate the required analyte from the other constituents. In these cases, the pretreatment unit may comprise a surface coated with an antibody against the analyte which is measured by a standard photometric method.

[0092] On the other hand, pretreatment may be used to purify the sample by immobilizing a cross-reacting substance using an antibody against the substance that is coated on the pretreatment surface. The purified sample can then be measured by standard turbidimetry or other methods.

[0093] Within some immunoassays antibodies may cross-react with substances related to the desired analyte. Pretreatment to remove such cross-reactants may avoid such problems and antibodies or other binding or marker substances with lower specificity may be used. Such an approach may lead to reductions of the costs of antibody development.

[0094] An important application of the invention are assays that today cannot be performed by photometric methods because of their low sensitivity. For instance, the preferred hormone to diagnose thyroid disorders, thyrotrophin-stimulating hormone (TSH), is typically so low in serum concentration that complicated techniques usually performed by separate instruments are used for measurements of TSH. If, however, an immunoaffinity pretreatment method for TSH is used to concentrate TSH approximately 10-fold or more, a useful turbidimetric assay is possible.

[0095] For food safety, analysis of various toxic mycotoxins in agricultural products is very important. For concentration and separation from sample matrices, extractions and immunoaffinity steps are necessary prior to assay. These steps may be automated by using specific antibodies or mycotoxin group binders for solid phase extraction in the pretreatment unit.

[0096] A similar approach is useful in environmental analysis when measuring cyanotoxins, produced by Cyanobacteria.

[0097] In the brewery industry, certain iso-alpha-acids are monitored during the brewing process to estimate bitterness of beer. The reference method used employs extraction with iso-octane. This procedure may be omitted and the assay automated by using pretreatment where the target iso-alpha-acids are bound to a surface and then eluted by a suitable solvent, to be transferred to measurement unit.

[0098] Many drugs of abuse (DoA) are tested in urine samples. This testing is frequently hampered by analyte related molecules or drug metabolites that cross-react with the employed antibodies, causing false positive results. With a suitable pretreatment the interfering molecules can be removed and a reliable assay performed thereafter.

[0099] On the other hand, many DoA assay suffer from lack of sensitivity which may lead to false negative results. Here, the urine sample can be directed into a column where drug molecules are retained, and then eluted with a smaller volume than the original samples. Thus better sensitivity and higher reliability is achieved.

[0100] One aspect of the invention is to enrich, purify or fractionate one or more analytes to be measured from a sample by retaining methods like immobilization, sieving or filtering. Immobilization may be done on surfaces or by antibodies so that desired substances or matrix components are retained. Filtering, for example by membranes, may be used to separate and retain molecules of desired size. Cell separation can be performed by flow-through microchannels. Pretreatment may be divided or arranged in two separate phases including pretreatments such as: reagent addition+incubation to form complex, reagent addition+incubation with proteolytic or other enzymes to be removed later, removal of lipids (lipemia) and removal of hemoglobin or other interfering substances. In one embodiment, the substance or substances that are to be measured are not chemically or physically reacted, modified or altered so, that the information content of a small sample used in discrete analysis is maintained as unchanged as possible.

[0101] Alternatively, the invention is based on picking desired substances from the sample in order to enrich the sample itself or, preferably the analyte to be measured directly. Different substance may require different pretreatment means, but the apparatus may be designed so that the pretreatment means such as columns can be easily changed according to assaying needs. The columns or other means may be disposable or reusable, and changed automatically.

[0102] In a discrete analysis several measurements may be done from a single sample. To accomplish this using the present invention, a sub-sample is removed from the primary sample and the pretreatment is done on the subsample, leaving the primary sample unchanged. Thus, the primary sample may be used for further analyses of other substances.

[0103] One aspect of the invention is to combine above described pretreatment methods with a discrete analysis apparatus. The idea is that pretreatment step operates integrated with the analyzer so that pretreatment can be performed only when the analyzer receives such a test request that includes a pretreatment step. The pretreatment step operates independently from the analyzer so that normal analyzing processes can be done simultaneously with the pretreatment. The analyzer performs requested measurements for each sample or subsample (sample aliquot) regardless whether the sample/subsample has been pretreated or not. Also, some of the subsamples of one sample analyte may be pretreated and some not, exactly as required. As the pretreatment step does not interfere with the measurements performed by the analyzer, several measurement cycles may occur simultaneously in the analyzer.

[0104] According to one valuable aspect of the invention, the invention utilizes a discrete fluid analyzing apparatus, wherein measurements are done photometrically or electrochemically on samples placed in separate wells. Reactants are used as needed to induce changes in the analyte in order to obtain response that can be measured photo-metrically or electrochemically. This type of measurement system is relatively inexpensive. Therefore, inexpensive ways to handle the analyte samples and subsamples are preferably used. For example, washable dispensers are used instead of disposable ones. This limits the invention to measurements wherein contamination, cross-contamination or mixing of samples can be avoided to a level that does not interfere with the measurements by washing and cleaning the dispensers or other fluid transfer means, such as dispensing needles, probes or washable pipette tips.

[0105] The sample or a subsample taken from it undergoes changes during measurement step when reactants are used. Therefore, the molecule (analyte) to be measured must not change chemically under the pretreatment step. This is a requirement for the pretreatment step to operate properly with a discrete analyzer.

[0106] FIG. 3 shows an example of some workflows that can be accomplished by the invention. First, a sample 1 is obtained. The sample may be any liquid sample, for example beer, wort, blood or urine. The sample is identified by a data carrier such as a RFID chip or bar code and when the sample vessel enters the analyzer, data is read, or the sample can be identified to the system manually by the user, the sample is identified and the operating system of the analyzer requests all pretreatments and measurements that are to be performed on the sample. The sample may be placed on queue or taken directly forwards. Now the whole sample or more commonly one or more subsamples are taken from the sample by a metallic needle and the extracted liquid is placed on measurement wells, such as cuvettes.

[0107] If pretreatment is needed, sample/subsample (an aliquot of the sample or subsample) is transferred to pretreatment step and pretreatment is performed as earlier has been described. In short, pretreatment includes capturing and releasing molecule that is to be measured in order to purify or enrich it. See the description of pretreatment process for further details. Pretreatment should not change the chemical composition of the analyte. The pretreated sample is transferred to a measurement step. Some examples are shown in FIG. 3. In Application 1 the sample (herein a whole sample, alternatively a subsample) can be measured photometrically without any chemical reaction. In this example (ref * in figure), pretreatment is part of the Application and is automatically generated by requesting the Application 1. In practice this application consists of pretreatment step with flexibility to design pretreatment parameters and the actual measurement step. In this case measurement is performed with no reagent additions. Application 1 (ref **) can be for example beer/wort bitterness or polyphenol analysis.

[0108] Applications 2 and 3 include dispensing at least sample aliquot (subsample), dispensing reagent, incubation and photometric analysis. In Application 3, two or more reagents are used (reagents 2, 3, 4, and 5).

[0109] When pretreatment step is not needed, sample, or an aliquot of the sample or subsample, can be processed according to Applications 4-7. Now the sample/subsample (aliquot of the sample or subsample) is measured photometrically without reacting with a reagent (Application 4), measured electrochemically without adding reagent (Application 5), or by similar methods as in Applications 2 and 3 (Applications 6 and 7). The Applications shown here are examples only and any measurement process application available in analyzer may be used in conjunction with both pretreatment steps, i.e. together with actual pretreatment or directing the sample or subsample directly to measurement step.

[0110] Applications 2-7 can be for example Beta-Glucan,

[0111] Glucose, pH, conductivity, hemoglobin, HbA1C analysis,

[0112] Ammonia or sulphate measurements.

[0113] Several samples, and their replicates, can be assayed simultaneously with several applications.

[0114] Sample dispensing does not require disposable tips. Vice versa, disposable tips should be avoided in order to keep the operating costs at bay. Application examples can have flexibility for volumes, dispensing orders and induce e.g additional sample blankings.

[0115] FIG. 4 shows one operating principle of the invention using an explanatory display template. Herein the process starts when client requests a test. This request can be delivered to the analyzer by a memory device or made manually when a sample is loaded to the analyzer. When a test is requested for a named sample, the analyzer automatically selects a pretreatment step including pretreatment or no pretreatment according to the specifications of the test as well as measurements, reagents, incubation time, mixing and other features. All parameters may be determined flexibly.

[0116] The left side of the display shows a list of test and treatments available. Herein a test Bitter 300 is requested (highlighted). The list shows that the test is photometric and pretreatment is on use. On the right side is information about the test which shows that pretreatment method is Bitter AU. Further details about reagents, samples, incubation and such used in a particular test are placed under tabs with same nominations (now shown in this display). The flow of the test is displayed on the right side of the tabs.

[0117] As can be seen on the display, Bitter 300 request performs automatically a Bitter AU pretreatment (selection: Pretreatment procedure: Bitter AU) and thereafter a photometric test typical to a discrete analyzer. The parameters of the photometric test are placed under the tabs bearing nominations of the parameters.

[0118] Multiple tests may be performed on one sample. As can be seen on the left side of the display, the analyzer is capable of performing multiple different tests (see test name) simultaneously and some of the tests may include pretreatment and some not. A pretreatment step can also be performed without a following photometric test requiring additional reagents. Usually a subsample of the original or pretreated sample is needed for each test.

[0119] Various embodiments of the method according to the invention are capable of performing optional, on demand pretreatment, simultaneous measurement and pretreatment, and/or measurement. Pretreatment may be performed on a whole sample r subsample or sample aliquot of the whole sample. Several tests may be performed on a single sample. Sample or sample aliquot can also be automatically treated or e.g. adjusted for pH by reagent addition before pretreatment.

[0120] Numerous embodiments of the present invention have been shown and described with attention paid to fundamental novel features of the invention as applied to the embodiments. However, it is understood that various omissions and substitutions and changes in the form and details of the invention may be made by those skilled in the art without departing from the spirit of the invention. For example, it is expressly intended that all combinations of those elements which perform substantially the same results are within the scope of the invention. Substitutions of the elements from one described embodiment to another are also fully intended and contemplated. It is also to be understood that the drawings are not necessarily drawn to scale but they are merely conceptual in nature. It is the intention, therefore, to be limited only as indicated by the scope of the claims appended hereto.