Abstract: The present invention discloses a test device including a support member; a top member mounted to the support member, wherein in the support member and the top member are configured such that they together define a discrete chamber. The discrete chamber includes a first well region including at least one first well; a second well region including at least one second well; and a channel region including at least one channel connecting the first well region and the second well region with one another. The second region is preferably horizontally offset with respect to the first well region in a test orientation of the device.

Abstract: The present invention recognizes that diagnosis and prognosis of many conditions can depend on the enrichment of rare cells from a complex fluid sample. In particular, the enrichment of fetal cells from maternal samples, such as maternal blood samples, can greatly aid in the detection of fetal abnormalities or a variety of genetic conditions. In addition, the present invention recognizes that the enrichment of rare malignant cells from patient samples, can aid in diagnosis, prognosis, and development of therapeutic modalities for patients. The invention includes microfabricated filters for filtering fluid samples and methods of enriching rare cells of fluid samples using microfabricated filters of the present invention. The invention also includes solutions for the selective sedimentation of red blood cells (RBCs) from a blood sample and methods of using selective RBC sedimentation solutions for enriching rare cells of a fluid sample.

Abstract: This invention provides methods of retentate chromatography for resolving analytes in a sample. The methods involve adsorbing the analytes to a substrate under a plurality of different selectivity conditions, and detecting the analytes retained on the substrate by desorption spectrometry. The methods are useful in biology and medicine, including clinical diagnostics and drug discovery.

Abstract: This invention provides methods of retentate chromatography for resolving analytes in a sample. The methods involve adsorbing the analytes to a substrate under a plurality of different selectivity conditions, and detecting the analytes retained on the substrate by desorption spectrometry. The methods are useful in biology and medicine, including clinical diagnostics and drug discovery.

Abstract: After various sugar nucleotide solutions and glycosyltransferases (or primers) have been mixed, they are introduced into a reaction tank (column) with primers (or glycosyltransferases) immobilized thereon. Then solutions coming out of the reaction tank are led to an ultrafiltration column. The oligosaccharide synthesizer according to the present invention is equipped with a flow path for ensuring that glycosyltransferases or primers separated by the ultrafiltration column are returned into a container for storing each solution in a sample injector.

Abstract: The present invention relates to protein separation systems and methods capable of resolving and characterizing large numbers of cellular proteins. In particular, the present invention provides a novel mass mapping system and methods for the differential display of proteins. The present invention further provides novel methods for displaying differential protein expression between two samples. In particular, the present invention provides novel method of mapping differential expression of proteins in non-cancerous, pre-cancerous, and cancerous cells.

Abstract: The present invention aims at providing a well unit to be used in fabricating an apparatus whereby movements of cells based on their own actions can be accurately and easily detected, in detecting the chemotaxis of cells due to a chemotactic factor or the inhibition of the chemotaxis of cells by an inhibitor. Accordingly, the present invention provides a well unit to be used in an apparatus for detecting chemotaxis of cells and separating cells characterized in that a plural number of wells, in which a liquid sample can be held in a resting state, are connected to each other via a channel, the channel is provided with a bank, and, in the upper part of the bank, barriers constituting one or more grooves having a width and/or a depth fit for the diameter or deformability of cells are provided or a plane is provided so as to give a gap having a depth fit for the diameter or deformability of cells between the plane and the glass substrate.

Abstract: This new automatic bio-manipulation factory system for manipulating a single cell comprises: a bio-MEMS that transports, separates, and extracts a single cell for manipulation from a plurality of cells, controls the rotation-orientation of the extracted cell automatically, and enables the extracted cell to maintain a certain temperature; and a micro manipulation system that obtains vision information and manipulation information about the extracted single cell and the manipulation tool, provides such information to operator, manipulates the extracted single cell by transmitting the rotation-orientation feedback information and the cell manipulation order of the operator to the bio-MEMS, and manipulates the manipulation tool by transmitting the tool manipulation order of the operator to the manipulation tool. The conventional manual and individualized single cell manipulation can be automated and processed collectively by systematically combining the bio-MEMS with the micro manipulation system.

Abstract: The invention comprises an apparatus and a method of effecting localized electroporation in a relatively small target area and for introducing foreign matter into cellular material in which the target area is located. The cellular material may be in vitro, in ovo or in vivo.

Abstract: The present invention provides methods for the purification of plasmid DNA that includes removal of host cell impurities, such as endotoxins, RNA, proteins, and chromosomal DNA, from an aqueous solution containing plasmid DNA and methods for separation and purification of supercoiled plasmid DNA from an aqueous solution containing a mixture of supercoiled and nicked or relaxed plasmid DNA using hydrophobic interaction chromatography supports.

Abstract: An instrument for conducting nucleic acid amplification reactions in a disposable test device. The test device includes a first reaction chamber containing a first nucleic acid amplification reagent (e.g., primers and nucleotides) and a second reaction chamber either containing, or in fluid communication, with a second nucleic acid amplification reagent (e.g., an amplification enzyme such as RT). The instrument includes a support structure receiving the test device. A temperature control system maintains the first reaction chamber at a first elevated temperature but simultaneously maintains the second nucleic acid amplification reagent at a second temperature lower than the first temperature so as to preserve the second nucleic acid amplification reagent. An actuator operates on a fluid conduit in the test device to place the first and second reaction chambers in fluid communication with each other after a reaction has occurred in the first reaction chamber at the first temperature.

Abstract: The present invention recognizes that diagnosis and prognosis of many conditions can depend on the enrichment of rare cells from a complex fluid sample. In particular, the enrichment of fetal cells from maternal samples, such as maternal blood samples, can greatly aid in the detection of fetal abnormalities or a variety of genetic conditions. In addition, the present invention recognizes that the enrichment of rare malignant cells from patient samples, can aid in diagnosis, prognosis, and development of therapeutic modalities for patients. A first aspect of the present invention is a microfabricated filter for filtering a fluid sample. A microfabricated filter of the present invention comprises at least one tapered pore, and preferably comprises at least two tapered pores whose variation in size is 20% or less. The present invention also includes a method of enriching rare cells of a fluid sample using a microfabricated filter of the present invention.

Abstract: In a bio-separation system, incident radiation (e.g., from a laser or LED source) for detection of separated analytes is directed at the detection zone axially along the separation medium, instead of through the boundary walls of the detection zone. In one embodiment, incident radiation at one or more wavelengths is directed via at least one optic fiber that extends axially along the separation medium to the proximity of the detection zone. Emitted radiation from the detection zone passes through the boundary walls about the detection zone for off-column detection, and/or is directed axially along the separation medium for on-column detection. In another aspect of the present invention, the detection zone is located at a widened zone along the separation channel. In a further aspect of the present invention, the optical detection configuration may be scaled up and implemented in a multi-channel CE system that comprises multiple capillary separation channels.

Abstract: In a bio-separation system, emitted radiation signals representative of sample analytes are collected from the detection zone axially along the separation medium, instead of through the boundary walls of the detection zone or the separation column. In one embodiment, emitted signals are collected via an optic fiber that extends from the proximity of the detection zone along the detection collar. According to another embodiment, a single dual purpose (excitation and emission) fiber or dual fibers (one for excitation radiation and the other for emitted radiation detection) are incorporated into detection collar. In another aspect of the present invention, the detection zone is located at a widened zone along the separation channel. In a further aspect of the present invention, the optical detection configuration may be scaled up and implemented in a multi-channel CE system that comprises multiple capillary separation channels.

Abstract: There are disclosed a stable factor VIII/vWF-complex, particularly comprising high-molecular vWF multimers, being free from low-molecular vWF molecules and from proteolytic vWF degradation products, as well as a method of producing this complex.

Abstract: An analyte is separated from a fluid sample by introducing the sample into a cartridge having an extraction chamber containing capture material for capturing the analyte. The sample is forced to flow through the extraction chamber to capture the analyte with the capture material in the extraction chamber. The captured analyte is then eluted from the extraction chamber by forcing an elution fluid to flow through the extraction chamber. The cartridge may optionally include a lysing region for lysing sample components (e.g., cells spores, or microorganisms), a waste chamber for storing waste fluid, and reaction or detection chambers for chemically reacting or detecting the eluted analyte.

Abstract: A device for lysing components (e.g., cells, spores, or microorganisms) of a fluid sample comprises a cartridge having a lysing chamber for receiving the sample and having at least one solid phase in the lysing chamber for capturing the sample components to be lysed. An ultrasonic transducer is coupled to a wall of the lysing chamber to transfer ultrasonic energy to the captured sample components.

Abstract: A device for use with an ultrasonic transducer to lyse components of a fluid sample comprises a cartridge having a lysing chamber, an inlet port in fluid communication with the lysing chamber, and an outlet port for exit of the sample from the lysing chamber. The inlet and outlet ports are positioned to permit flow of the sample through the lysing chamber, and the chamber contains at least one solid phase for capturing the sample components to be lysed as the sample flows through the chamber. The lysing chamber is defined by at least one wall having an external surface for contacting the transducer to effect the transfer of ultrasonic energy to the chamber.

Abstract: A method of injecting, isolating and separating mixed analytes from one or more samples. The method includes collecting successive fractions from each of a plurality of samples at discrete points in time. Fractions may be analyzed at the time of collecting, or later, using one or more detector systems. In one embodiment, a processor controls the elutions of fractions by modulating the migration field in a separation pathway. The processor also controls distribution of the fraction into a particular collection well of a plurality of collection wells.

Abstract: Devices are provided which include supports upon which one or more ion-exchange materials can be disposed for purifying a sample. In various embodiments, the supports include a plurality of deformable members, for example, petal-shaped purification members, that provide binding sites for ion-exchange material and optionally biochemical species, chemicals, salts, or other materials. An apparatus and method are also provided for the insertion and removal of the purification members into respective wells of a multi-well microplate.

Abstract: Microfluidic devices are provided having units of 8-fold symmetry comprising 8 assay units, where a reservoir provides a common component to 8 assay units. The units can be compactly formed in a substrate to provide the ability to perform a large number of assays within a small area. The microfluidic devices find use in operations, such as assays, DNA sequence detection, etc. Various formats can be used to have the microfluidic device interrelate with microtiter well plates. Methods are provided for monitoring the flow rates/velocities of assay components and streams for comparison of results in different assay units and/or to modify the conditions to change the flow rates in particular channels.

Abstract: A method of separating a cell-containing sample into a substantially cell-depleted portion, and a cell-containing portion comprising at least one of a stem cell, a lymphocyte, and a leukocyte comprises a step in which the sample is received in a vessel with at least one flexible wall. In another step, an additive and particles are added to the sample, wherein the additive substantially binds to the at least one of the stem cell, lymphocyte, and leukocyte, and the particles and wherein the particles substantially bind to the at least one of the stem cell, lymphocyte, and leukocyte, and the additive, thereby producing a cell-containing network. In a further step, the network is separated from the substantially cell-depleted portion by applying a magnetic force.

Abstract: This invention provides methods of retentate chromatography for resolving analytes in a sample. The methods involve adsorbing the analytes to a substrate under a plurality of different selectivity conditions, and detecting the analytes retained on the substrate by desorption spectrometry. The methods are useful in biology and medicine, including clinical diagnostics and drug discovery.

Abstract: A sampling device for detecting airborne particles has two parallel substantially identical intake passages. Each intake passage, in close proximity to the other, and has an inlet, a sampling filter intersecting the passage, means for drawing ambient air through the inlet, and sampling filter. The sampling filter is mounted on a filter magazine that has a plurality of sampling filters. A motor is provided for sequentially moving successive sampling filters on the magazine into the air flow. The sampling device accordingly captures two substantially identical samples from the parallel intake passages.

Abstract: An apparatus, compositions and related methods for sequencing a target nucleic acid are described. In certain embodiments, the apparatus is a microfluidic apparatus comprising an input chamber, microchannel, output chamber and a detection unit that is operatively connected to the microchannel. In preferred embodiments, the methods include hybridizing a target nucleic acid to one or more probe libraries, moving the hybridized target nucleic acid past the detector, and detecting bound probes. Probe libraries may comprise oligonucleotides or oligonucleotide analogs, preferably with each probe uniquely labeled. A linear order of labeled probes hybridized to the target nucleic acid can be detected and the target nucleic acid sequence deduced. In preferred embodiments, probe labels are detected by analysis of electron-induced fluorescence of probes labeled with conductive polymers.

Abstract: Arrays of flowable or fixed particle sets are used in microfluidic systems for performing assays and modifying hydrodynamic flow. Also provided are assays utilizing flowable or fixed particle sets within a microfluidic system, as well as kits, apparatus and integrated systems comprising arrays and array members.

Abstract: The present invention allows the determination of trace levels of ionic substances in a sample solution (ions, metal ions, and other electrically charged molecules) by coupling a separation method, such as liquid chromatography, with ion selective electrodes (ISE) prepared so as to allow detection at activities below 10−6M. The separation method distributes constituent molecules into fractions due to unique chemical and physical properties, such as charge, hydrophobicity, specific binding interactions, or movement in an electrical field. The separated fractions are detected by means of the ISE(s). These ISEs can be used singly or in an array. Accordingly, modifications in the ISEs are used to permit detection of low activities, specifically, below 10−6M, by using low activities of the primary analyte (the molecular species which is specifically detected) in the inner filling solution of the ISE. Arrays constructed in various ways allow flow-through sensing for multiple ions.

Abstract: A method and apparatus are provided for the separation of biological materials from one another. The device includes a separation column that is in flow communication with a collection chamber in a reservoir that includes a collection container and a lid. The separation column is mounted on the lid. The collection container includes a concentration zone at the bottom and the container is adapted for use in further separation steps including centrifuging. The device includes a connector that is in flow communication with the collection chamber and is adapted for connecting it to a source of vacuum to help induce flow of liquid through the separation column.

Abstract: A method and device is disclosed for rapidly identifying a large number of proteins by placing a protein mixture in a sample chamber of an electrophoresis gel, and performing electrophoresis to separate the mixture by molecular weight, in a direction of separation, into a two-dimensional separation pattern in the gel. The separation pattern is transferred to a membrane, such as a sheet of nitrocellulose, and a plate with a set of separate, side-by-side slots is then applied to the membrane. A different antibody mixture is introduced into each of the slots by perfusing each antibody mixture under pressure through the slots. The antibody mixture that is perfused through each slot recognizes several different proteins of sufficiently different molecular weights that different protein bands can be resolved by the antibody mixture in each slot.

Abstract: A device for separating an analyte from a fluid sample comprises a cartridge incorporating a flow-through microfluidic chip. The microfluidic chip includes an extraction chamber having an array of microstructures for capturing the analyte and for subsequently releasing the captured analyte into an elution fluid. Each of the microstructures has an aspect ratio of at least 2:1. The cartridge also includes channels and at least one low controller (e.g., one or more valves) for directing the flow of the sample and elution fluid through the microfluidic chip. The cartridge may optionally include a lysing region for lysing sample components (e.g., cells spores, or microorganisms), a waste chamber for storing waste fluid, and reaction or detection chambers for amplifying or detecting the analyte.

Abstract: The present invention provides, in one aspect, an apparatus for electrophoretic separation of analytes. In one aspect, the apparatus comprises a disc-shaped substrate defining (1) a central reservoir region, (2) a plurality of electrophoretic channels in fluid communication with, and emanating substantially radially from, the central reservoir region, the channels being coplanar with each other, and each channel having (i) a proximal end which is linked to the reservoir region, and (ii) a distal end, and preferably (3) for each channel, at least one chamber, and preferably three chambers, linked by a passageway in fluid communication with the distal end of that channel.

Abstract: A method and apparatus for extracting, identifying, and manipulating proteins or peptides from a solution uses paramagnetic beads having a coating with an affinity for the target component. In one embodiment, paramagnetic beads coated with C18 are used to adsorb proteins and peptides. The beads can be used to purify, immobilize and assay antibodies. By cycling the beads, many times greater molar amount of binding partner may be separated from a solution. A magnetic probe is used to capture the beads and transfer the beads to selected processing stages.

Abstract: An immunoassay of a liquid biological specimen for a specific analyte is conducted by forming a test sample by contacting the specimen with: (1) a binding substance of a ligand, antiligand or receptor capable of binding the analyte and (2) a detector substance of a colloidal gold or silver labeled ligand or antiligand to form a test sample. The test sample is applied by flowing onto a defined zone of an insoluble porous support film having a pore size impassable to a complex formed between the analyte, if present, with the binding substance and the detector substance, but passable to the binding substance and detector substance while remaining uncomplexed in the absence of the desired analyte. If the analyte is present in the test specimen, the detector substance binds with the analyte and the binding substance to form a visually inspectable complex on the surface of the porous support film.

Abstract: A method for collecting a microbiological substance utilizes a microfabricated biochip having a collection chamber. A fluid sample containing a microbiological entity of interest is delivered to the collection chamber in the biochip. Then a non-uniform electric field is generated in the collection chamber, to retain the microbiological entity of interest in the collection chamber. The microbiological entity is retained through dielectrophoresis induced by the energization of the electrodes by a periodically applied, alternating current.

Abstract: An assay assembly comprising a chip on which an array of reactive species is immobilized, the chip being located in a storage well having a base and side walls.

Abstract: A method and apparatus for monitoring a microbial material in a fluid sample, the method includes, providing a fluid sample for microbiological analysis, and optionally selectively permitting multiplication of microbial material present in the fluid sample. The microbial sample is then permitted to enter a reaction chamber containing at least one capture member arranged to selectively capture the multiplied microbial material thereon and optionally washing the capture member having the microbial material captured thereon. The amount of the captured microbial material present on the capture member is subsequently monitored.

Abstract: A method for purifying factor VIII/vWF complex or free vWF by immunoaffinity chromatography in a form suitable for use as a medicament. Factor VIII/vWF complex or free vWF is recovered from an immunoaffinity adsorbent by using an eluting agent containing a zwitterionic species. The presence of the zwitterionic species allows for the use of mild conditions throughout the preparation, facilitating retention of molecular integrity, activity, and incorporation of the recovered proteins into pharmaceutical preparations without the need for additional stabilizers or preservatives.

Abstract: The present invention relates to a filtration-detection device for detecting or quantifying an analyte in a test sample including a filtration device having a first binding material immobilized thereto, wherein the first binding material is capable of binding to a portion of the analyte, and a detection assembly positioned relative to the filtration device to detect or quantify analyte bound to the first binding material. The present invention also relates to methods of using the filtration-detection device.

Abstract: A process and device are disclosed to determine the activity of enzymes in liquids in a largely automatic manner. The device for carrying out this process has a column (1) with a chromatographic carrier for treating a measurement sample. The carrier is mixed with a substance capable of binding to an enzyme inhibitor present in the measurement sample and that corresponds to at least one enzyme. A measurement sample supply (2) is associated to one end of the column (1). A valve/pump arrangement (7, 11, 14, 15) for filling at least one test tube (5) with a carrier and at least part of the measurement sample is connected downstream of the column (1), in the flow direction of the measurement sample. The carrier is dissociated into cleavage products by the action of the enzyme. The rise in concentration per unit of time of at least one of the cleavage products of the carrier is sensed during an incubation time.

Abstract: The present invention concerns a method for rapidly identifying biological agents in a sample suspected of containing the same. The biomarkers are released from biological agents present in a sample, separated from contaminants present in the sample, ionized to form an ionized stream of biomarkers which are sent to a mass spectrometer to obtain a mass spectral profile of the biomarkers in the sample for analysis and identification. The present invention is also directed to an apparatus useful for carrying out the steps of the above method in an automated mode.

Abstract: Disorders are diagnosed by analyzing biological samples of ad libitum-fed and dietary-restricted individuals to generate frequency distribution patterns representative of molecular constituents of the samples, and comparing the patterns.

Abstract: A biological fluid collection container comprising a cup member, a lid assembly removably mounted to the cup member comprising a housing with a downwardly extending cylindrical skirt, a luer lock with a throughgoing bore extending from one side of the lid housing. A hollow tube extends from the other side of the lid housing adjacent the luer lock and is axially aligned with the throughgoing bore of the luer lock. The hollow tube is provided with a plurality of throughgoing holes leading into its lumen along its surface to provide for a sampling along various liquid level layers of the biological fluid specimen collected in the cup member so that when the biological fluid specimen is removed from the cup member a representative sampling is obtained.

Abstract: The invention relates to a microfluidic system (110), particularly a microfluidic chip, with at least one operational channel (170), in which a fluid and/or the constituents contained therein are moveable in the direction of the operational channel (170) by a driving force, particularly by using pressure, acoustic energy, or an electrical and/or a magnetic field. At least one measurement sensor (185) used to measure a measurable value assigned to the fluid and derivable in the region of the fluid and at least one regulator (192) is provided to regulate the driving force and/or a parameter that may be influenced by it, wherein the regulator (192) is coupled with a measurement sensor (185) and a device used to alter the driving force (189) and/or the parameter that may be influenced by it. The invention further relates to a procedure to transport and guide a fluid and/or the constituents contained therein within a microfluidic system of the type described above.

Abstract: Apparatus and methods for modulating flow rates in microfluidic devices are provided. The methods involve modulating downstream pressure in the device to change the flow rate of materials in an upstream region of the device. Such methods include electrokinetic injection or withdrawal of materials through a side channel and the use of an absorbent material to induce wicking in the channel system. The apparatus provided includes a prefabricated wick in the device to provide for flow rate control. Additional methods for determining velocity of a particle and cell incubation time are also provided.

Abstract: An adapter plate transforms a vacuum manifold designed for standard multi-well plates that are commonly used in biochemical laboratories into one that can be used for columns such as chromatographic columns, filtration columns, and the like. The plate contains an array of male-female-type complementary connectors, each connector fitted with a removable plug that seals the connector off when not in use. The plate thus allows the manifold to be used for as few as one column or any number up to the total number of connectors in the plate.

Abstract: High expression levels and simple downstream processing are essential for the production of recombinant proteins at low cost. We report here a new expression vector which allows production of fusion proteins in Dictyostelium discoideum. We made use of the ability of Discoidin I to bind Sepharose-4B to set up a nearly single step purification procedure. The Discoidin I coding region was fused to several forms of the malaria parasite CSP gene in a Dictyostelium expression vector, allowing intracellular accumulation as well as partial secretion via a pathway unrelated to the endoplasmic reticulum and Golgi. The fusion proteins present in cell extracts were affinity-purified over Sepharose-4B columns. Addition of a signal peptide allowed endoplasmic reticulum targeting and glycosylation of the fusion protein. Inclusion of a thrombin cleavage site allowed to cleave Discoidin from the CSP protein. The use of stable and low cost Sepharose 4B as affinity matrix should allow large-scale preparations.

Abstract: A device for transfer suitable for capturing and releasing microparticles binding an immobilized substance, which includes a magnet as well as either an extendable membrane, shapable membrane or magnet's coating such that the membrane or coating pressing tightly against the magnet's surface separates the magnet from the microparticles but does not substantially weaken the magnetic field directed at the microparticles. Also disclosed is a method for transferring a microparticle immobilized substance from a first vessel to a second vessel where the microparticles are of a magnetic or magnetizable material or the microparticles are attached to a magnetic or magnetizable body and the microparticles with the substance immobilized thereupon are captured with the aid of a magnet submerged in the first vessel, the magnet along with the microparticles captured thereupon are transferred to the second vessel and released from the magnet's influence.

Abstract: A method of injecting, isolating and separating mixed analytes from one or more samples. The method includes collecting successive fractions from each of a plurality of samples at discrete points in time. Fractions may be analyzed at the time of collecting, or later, using one or more detector systems. In one embodiment, a processor controls the elutions of fractions by modulating the migration field in a separation pathway. The processor also controls distribution of the fraction into a particular collection well of a plurality of collection wells.

Abstract: A cartridge (101) for separating a desired analyte from a fluid sample includes a sample port (103) and a sample flow path extending from the port through the body of the cartridge. The sample flow path includes at least one flow-through component (122), e.g., filter paper or a microfabricated chip, for capturing the desired analyte from the sample as the sample flows through the cartridge. The cartridge also includes an elution flow path for carrying elution fluid through the component (122) to release captured analyte from the component into the elution fluid. The elution flow path diverges from the sample flow path after passing through the component (122). Flow controllers (41A) and (41B) direct the remaining fluid sample into a waste chamber (139) after the sample flows through the component (122) and direct the elution fluid and eluted analyte into a reagent chamber (141) and reaction chamber (143).

Abstract: A biological fluid collection container comprising a cup member, a lid assembly removably mounted to the cup member comprising a housing with a downwardly extending cylindrical skirt, a luer lock with a throughgoing bore extending from one side of the lid housing. A hollow tube extends from the other side of the lid housing adjacent the luer lock and is axially aligned with the throughgoing bore of the luer lock. The hollow tube is provided with a plurality of throughgoing holes leading into its lumen along its surface to provide for a sampling along various liquid level layers of the biological fluid specimen collected in the cup member so that when the biological fluid specimen is removed from the cup member a representative sampling is obtained.