WO2011146596A1

WO2011146596A1 - Method and apparatus for performing extraction and enrichment of components in a biological sample

Info

Publication number: WO2011146596A1
Application number: PCT/US2011/036972
Authority: WO
Inventors: David Friedman; Rajiv Pande; Keld Sorensen
Original assignee: Cyvek, Inc.
Priority date: 2010-05-18
Filing date: 2011-05-18
Publication date: 2011-11-24

Abstract

A method and apparatus for processing a sample in a hollow element having a narrow interior channel functionalized with an enzyme, antibody or other affinity moiety selected in relation to a treatment of the sample; and providing a pre-treated sample from the narrow interior channel that has either a substantial enrichment and pre-concentration of the sample, or a depletion of an interfering substance in the sample, or an activation of a latent molecule in the sample, or a degradation of the sample so to allow a subsequent capture of a desired analyte of interest, or a relevant molecule, or a latent molecule, or a reactive moiety in a downstream further processing of the pre-treated sample. The sample may include a biological or patient sample, such as serum, plasma, cerebrospinal fluid, urine, blood, saliva, tears, lavage fluid, milk, water, cell / tissue culture supernatants, etc.

Description

METHOD AND APPARATUS FOR PERFORMING EXTRACTION

AND ENRICHMENT OF COMPONENTS IN A BIOLOGICAL SAMPLE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit to provisional patent application serial no.

61/345,787, filed 18 May 2010, which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1 . Field of the Invention

The present invention relates to a method and apparatus for performing assays; and more particularly relates to a method and apparatus for performing chemical, biological or biochemical assays using microfluidic technology.

2. Brief Description of Related Art

Biological samples frequently need to be purified, enriched, activated or depleted prior to being analyzed. This purification, concentration, activation or dilution of biological samples is performed in a variety of ways; most of these methods require an off-line process. The pre-treatment of a sample in a separate vessel, adds a level of complexity, is time consuming, increases the cost, and requires the addition of precisely measured reagents for execution. Typically, the type of sample treatment required depends on the analyte of interest, and this becomes a major problem during multiplexed analysis as all assays have to be subjected to an identical sample treatment process. In addition, this complicates the analytical process and extends the analysis time unnecessarily. Additionally, an analyte having an unusual sample preparation requirement, cannot be measured in a multiplex panel, and has to be assayed individually. Moreover, current methodology such as ELISA plates and Luminex assays all suffer from the

shortcoming that the assay takes place in a single well with all components present. This means that the addition of the processing component (e.g. enzyme) must be done by the operator, or alternatively, must be added by the manufacturer of the well (plates).

An example of the need for sample pre-treatment of the sample prior to analysis would be the detection of Transforming Growth Factor beta (TGF-B). The quantification of TGF-B is actively sought after in life sciences. This is especially so in the areas of inflammation research and cancer research. The measurement of TGF-B is complicated as it is present in platelet granules and is released upon platelet activation. Therefore, to accurately measure circulating levels of TGF-1 , the TGF-B has to be optimally activated. This requirement necessitates an offline activation of TGF-B, rendering it almost impossible to measure TGF-B in conjunction with other markers (that do not require an activation step).

SUMMARY OF THE INVENTION

The present invention employs and provides further applications for a novel reaction vessel that is disclosed in PCT patent application no. PCT/US10/57860, filed 23 November 2010, as well as PCT patent application no. PCT/US1 1/29736, filed 24 March 201 1 , which is a continuation in part of the earlier filed PCT

application, and which are both hereby incorporated by reference in their entirety. The aforementioned PCT applications are assigned to the assignee of the present PCT application.

The assignee's novel hollow element reaction vessel allows a sample to be channeled into different microfluidic vessels, with each path (containing a unique hollow element) capable of being subjected to a different set of conditions (or sample pre-treatment conditions) thus enabling multi-analyte output. Aside from being used in the platform disclosed in the aforementioned PCT applications, this hollow element reaction chamber also lends itself very well to being utilized in any microfluidic device, for a variety of sample pre-treatment applications. A few embodiments are set forth by way of example below:

Sample enrichment According to this embodiment of the present invention, a sample pre-treatment hollow element could contain an immobilized affinity molecule capable of binding a sample so as to extract the sample (e.g. a bacterium sub-class) from large sample volumes. Following this affinity capture, the sample pre-treatment hollow element would be subjected to a physico-chemical elution (change in pH or ionic strength) that would enable the release of, e.g., an isolated analyte (now concentrated) to allow its capture (and subsequent recognition) downstream by the capture hollow element.

Sample depletion: According to this embodiment of the present invention, a sample pre-treatment hollow element would contain an immobilized protein or a factor capable of removing an interfering substance so as to allow a downstream recognition of a "relevant" molecule by a subsequent capture hollow element reaction vessel. As an example, the removal of albumin may be necessary to measure a specific plasma component via a chemistry assay.

Sample activation: According to this embodiment of the present invention (TGF-B example), a sample pre-treatment hollow element would contain an immobilized protein or a factor capable of activating a latent molecule so as to allow its recognition and downstream capture by a subsequent capture hollow element reaction vessel. Sample degradation: According to this embodiment of the present invention, a sample pre-treatment hollow element would contain an immobilized enzyme capable of degrading the sample so as to allow access to a reactive moiety (which would be recognized and captured downstream by a subsequent capture hollow element reaction vessel).

According to some embodiments, the present invention provides a new and unique method and apparatus for processing a biological or patient sample (such as serum, plasma, cerebrospinal fluid, urine, blood, saliva, tears, lavage fluid, milk, water, cell / tissue culture supernatants, etc.).

According to some embodiments, the present invention may take the form of a method featuring processing a sample in at least one hollow element having at least one narrow interior channel functionalized with at least one enzyme, antibody or other affinity moiety selected in relation to a treatment of the sample; and providing a pre-treated sample from the at least one narrow interior channel that has either a substantial enrichment and pre-concentration of the sample, or a depletion of an interfering substance in the sample, or an activation of a latent molecule in the sample, or a degradation of the sample so to allow a subsequent capture of at least one desired analyte of interest, or a relevant molecule, or a latent molecule, or a reactive moiety in a downstream further processing of the pre-treated sample.

According to some embodiments, the present invention may take the form of apparatus, including a reaction vessel, featuring at least one hollow element having at least one narrow interior channel functionalized with at least one enzyme, antibody or other affinity moiety selected in relation to a treatment of a sample; the at least one hollow element configured to:

receive the sample, and provide a pre-treated sample from the at least one narrow interior channel that has either a substantial enrichment and pre-concentration of the sample, or a depletion of an interfering substance in the sample, or an activation of a latent molecule in the sample, or a degradation of the sample so to allow a subsequent capture of at least one desired analyte of interest, or a relevant molecule, or a latent molecule, or a reactive moiety in a downstream further processing of the pre-treated sample.

According to some embodiments, the present invention provides a method and apparatus for performing extraction and enrichment of components in the biological or patient sample by functionalizing interior channels of hollow elements or beads with antibodies or other biomolecules selected to retain specific analytes of interest. In this way, the biological or patient sample can be processed in the interior channels of hollow elements or beads, and desired analytes can be effectively extracted from, e.g., an entire blood sample, and can be released by, e.g., a simple pH elution. Once released, the analytes can be further processed, e.g., by being neutralized in order for their rebinding downstream in, e.g., a quantification section of an immunoassay instrument. This provides a substantial enrichment and pre- concentration of the sample such that analytes of interest are presented to an analysis chamber of the immunoassay instrument at a 10-50 fold higher

concentration as compared to traditional approaches known in the art, thus enabling low level antigen detections.

By way of example, according to some embodiments the present invention may take the form of a method comprising:

processing a sample in at least one hollow element having at least one narrow interior channel functionalized with at least one enzyme, antibody or other affinity moiety selected to concentrate at least one desired analyte of interest present in the sample; and

providing a substantial enrichment and pre-concentration of the sample from the at least one narrow interior channel so that the at least one desired analyte of interest may be further processed at a substantially higher concentration, including in a range of about a 10-50 fold higher concentration, thus enabling at least low level antigen detections in relation to the sample.

The further processing may include being presented to an analysis chamber of an immunoassay instrument, as well as being further processed by at least one subsequent hollow element having at least one subsequent narrow interior channel functionalized with at least one subsequent enzyme, antibody or other affinity moiety selected to further concentrate the at least one desired analyte of interest present in the substantial enrichment and pre-concentration of the sample.

Here, the at least one hollow element having the at least one narrow interior channel functionalized with the at least one enzyme, antibody or other affinity moiety selected to concentrate at least one desired analyte of interest present in the sample may take the form of a first reaction vessel.

According to some embodiments of the present, the substantially higher concentration, including in the range of about the 10-50 fold higher concentration, of the at least one desired analyte of interest may be based at least in whole, or in part, on the processing of the sample in one reaction vessel, as well as in a series of two or more reaction vessels consistent with that set forth herein.

According to some embodiments, the method may also include one or more of the following features: In the method, the at least one enzyme, antibody or other affinity moiety may be selected to retain the at least one desired analyte of interest in the at least one narrow interior channel.

The provisioning of the substantial enrichment and pre-concentration of the sample from the narrow interior channel, and the further processing of the

substantial enrichment and pre-concentration of the sample, may include one or more of the following steps:

releasing the at least one desired analyte of interest, including by a pH elution process;

neutralizing the at least one desired analyte of interest in order for its rebinding downstream, including in a quantification section of an

immunoassay instrument;

rebinding the at least one desired analyte of interest; and/or

analysing the at least one desired analyte of interest, including in the analysis chamber of the immunoassay instrument, to detect the at least low level antigen in relation to the sample.

The method may also include a step for providing the substantial enrichment and pre-concentration of the sample to the at least one subsequent hollow element having the at least one subsequent narrow interior channel functionalized with the at least one subsequent enzyme, antibody or other affinity moiety selected to further concentrate the at least one desired analyte of interest present in the substantial enrichment and pre-concentration of the sample.

Here, the at least one subsequent hollow element having the at least one subsequent narrow interior channel functionalized with the at one subsequent least one enzyme, antibody or other affinity moiety selected to concentrate the at least one desired analyte of interest present in the sample may take the form of a second or subsequent reaction vessel.

The method may also include a step for arranging the at least one hollow element and the at least one subsequent hollow element for in-line processing, including where the in-line processing includes an on-line selection or de-selection of the at least one analyte of interest to isolate its reactive element or component, including by treating the at least one analyte of interest to a series of enzymes to isolate bio-molecules of interest that may be subsequently captured or characterized for different antigens or factors using a series of hollow elements having

appropriately functionalized narrow interior channels with appropriate enzymes, antibodies or other affinity moieties.

The method may include one or more of the following as well: The at least one enzyme, antibody or other affinity moiety may release the at least one desired analyte of interest from the sample. The at least one enzyme, antibody or other affinity moiety may be an immobilized component that converts the at least one analyte of interest to another compound which is more easily measured. The at least one enzyme, antibody or other affinity moiety may comprise some combination of identical or different enzymes, antibodies or other affinity moieties. The at least one enzyme, antibody or other affinity moiety may remove or convert an interfering substance in the sample that would otherwise adversely affects the ability of the at least one analyte of interest to provide a true response when treated.

The method may also be characterized by the at least one narrow interior channel of the at least one hollow element having one or one of the following features: The at least one narrow interior channel of the at least one hollow element may be configured or designed with a cavity or chamber having a very small inside diameter that is approximately 10μηη inner diameter (ID)) and a length-to-I.D aspect ratio of approximately 20:1 , which is approximately 20Όμηη L. The at least one hollow element or the at least one subsequent hollow element may be configured or designed as a honeycomb structure having a multiplicity of cavities or chambers having a very small inside diameter that is approximately 10μιτι inner diameter (ID)).

According to some embodiments, the present invention may take the form of apparatus, including a reaction vessel, comprising:

at least one hollow element having at least one narrow interior channel functionalized with at least one enzyme, antibody or other affinity moiety selected to concentrate at least one desired analyte of interest present in a sample;

the at least one hollow element configured to:

receive the sample, and

provide a substantial enrichment and pre-concentration of the sample from the at least one narrow interior channel so that the at least one desired analyte of interest may be further processed at a substantially higher concentration, including in a range of about a 10-50 fold higher concentration, thus enabling at least low level antigen detections in relation to the sample. Consistent with that set forth above, the apparatus may comprise one or more further processing device, apparatus or equipment for further processing the substantial enrichment and pre-concentration of the sample, including an analysis chamber of an immunoassay instrument, as well as at least one subsequent reaction vessel including at least one subsequent hollow element having at least one subsequent narrow interior channel functionalized with at least one subsequent enzyme, antibody or other affinity moiety selected to further concentrate the at least one desired analyte of interest present in the substantial enrichment and pre- concentration of the sample. The at least one subsequent hollow element is configured to receive the substantial enrichment and pre-concentration of the sample, and provides a subsequent substantial enrichment and pre-concentration of the sample from the at least one subsequent narrow interior channel so that the at least one desired analyte of interest may be further processed, if desired.

According to some embodiment, the apparatus may include one or more of the features set forth above in relation to the aforementioned method, as well as the following features:

The apparatus may comprise at least one further processing device, apparatus or equipment configured to do one or more of the following:

release the at least one desired analyte of interest, including by a pH elution process;

neutralize the at least one desired analyte of interest in order for its rebinding downstream, including in a quantification section of an

immunoassay instrument;

rebind the at least one desired analyte of interest;

analyze the at least one desired analyte of interest, including in the analysis chamber of the immunoassay instrument, to detect the at least low level antigen in relation to the sample.

The at least one subsequent hollow element having the at least one subsequent narrow interior channel functionalized with the at least one subsequent enzyme, antibody or other affinity moiety selected to further concentrate the at least one desired analyte of interest present in the substantial enrichment and pre- concentration of the sample may be configured to receive the substantial enrichment and pre-concentration of the sample, and further process the same consistent with that set forth herein.

The apparatus may also comprise at least one hollow element and the at least one subsequent hollow element being configured for in-line processing, consistent with that set forth herein.

Advantages

According to the present invention, the inclusion of a processing reagent (such as an enzyme) in the flow of the assay has numerous advantages.

The primary benefit is that it makes it possible to measure analytes that need processing prior to being available for the immunoassay

- The cartridges can be manufactured with various amounts, i.e., they can be calibrated to the assay in question.

- The contact time can be easily established by the flow rate.

- Several enzymes can be used in sequence.

- The system can either enrich the analyte for superior signal generation in

subsequent GNRs or it can remove interfering substances.

Moreover, the novel reaction vessel disclosed in the aforementioned PCT applications, in and of itself, enables very low cost manufacturing, fast reaction time, low sample volume, high sensitivity, and large dynamic range. The novel hollow reaction vessel may take the form of the at least one hollow element that has been functionalized with the capture moiety or capture molecules. By way of example, the at least one hollow element may be made from optical fiber so as to form hollow glass cylinder. Additional advantages of this novel reaction vessel include

substantially minimizing the need to design around cross reactivity by providing a means for automatically separating components where negative cross reactions occur.

As disclosed in the aforementioned PCT applications, additionally this unique assay device improves ease of use by employing a disposable microfluidic assay cartridge that will automate some of the manual steps typically associated with these types of tests. This assay device optimizes buffer conditions to produce

independently optimized assays. The optimized buffer conditions may include optimizing in relation to the pH, salinity or both. This assay device also allows samples to be independently diluted with buffer solution with respect to each channel.

BRIEF DESCRIPTION OF THE DRAWING

The drawing, which are not necessarily drawn to scale, includes the following Figures:

Figure 1 , which includes Figures 1 a, 1 b, 1 c, 1d and 1 e, shows block diagrams of methods for implementing some embodiments of the present invention, where Figure 1 a is a block diagram of a method for processing a sample according to some embodiments of the present invention, where Figure 1 b is a block diagram of a method for further processing the sample in relation to the basic method shown in Figure 1 a according to some embodiments of the present invention, where Figure 1 c is a block diagram of a method for further processing the sample in relation to the basic method shown in Figure 1 a according to some embodiments of the present invention, where Figure 1 d is a diagram of a hollow element having a narrow interior channel functionalized with at least one enzyme, antibody or other affinity moiety configured to process a sample according to some embodiments of the present invention, and Figure 1 e is a block diagram of a basic method for processing a sample according to some embodiments of the present invention,.

Figure 2, which includes Figures 2a, 2b, 2c and 2d, shows block diagrams of apparatus for implementing some embodiments of the present invention, where Figure 2a is a block diagram of apparatus for processing a sample according to some embodiments of the present invention, where Figure 2b is a block diagram of apparatus for further processing the sample in relation to the basic apparatus shown in Figure 2a according to some embodiments of the present invention, where Figure 2c is a block diagram of a method for further processing the sample in relation to the basic method shown in Figure 2a according to some embodiments of the present invention, and where Figure 2d is a block diagram of the basic apparatus for processing a sample according to some embodiments of the present invention,.

Figure 3, which includes Figures 3a, 3b, shows block diagrams of apparatus for implementing some embodiments of the present invention, where Figure 3a is a block diagram of apparatus for processing and further processing a sample using a series of enzymes treatments in conjunction with a subsequent series of antibody treatments related to the methods shown in Figure 1 and the apparatus shown in Figure 2 according to some embodiments of the present invention, and where Figure 3b is a block diagram of apparatus for processing and further processing a sample using an alternating series of enzymes and antibody treatments related to the methods shown in Figure 1 and the apparatus shown in Figure 2 according to some embodiments of the present invention.

Figure 4, which includes Figures 4a, 4b and 4c, where Figure 4a is a diagram of a sample enrichment pre-column for processing a sample according to some embodiments of the present invention, where Figure 4b is a diagram showing the processing of a sample using blood cell exclusion from blood according to some embodiments of the present invention, and where Figure 4c is a diagram showing the processing of a sample where plasma enters pores or otherwise narrow interior channels according to some embodiments of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Figure 1

By way of example, Figure 1 a shows the basic method generally indicated as 10, according to some embodiments the present invention, which includes two steps 12 and 14. The first step 12 features processing a sample 16 (see Figures 1 d and 3a) in at least one reaction vessel 40 including at least one hollow element 42 (see Figures 2a, 3a, 3b) having at least one narrow interior channel 44 (see Figures 2a, 3a) functionalized with at least one enzyme, antibody or other affinity moiety generally indicated by way of example as label 42a (see Figure 1 d) selected to concentrate at least one desired analyte of interest generally indicated by way of example as label Ag (representing, e.g., a captured antigen) present in the sample 16. The second step 14 features providing a substantial enrichment and pre- concentration of the sample generally indicated by the arrow 18 (see Figures from the at least one narrow interior channel 44 so that the at least one desired analyte of interest Ag (Figure 1 d) may be further processed at a substantially higher

concentration, including in a range of about a 10-50 fold higher concentration, thus enabling at least low level antigen detections in relation to the sample.

By way of example, the sample 16 may include a biological or patient sample, such as serum, plasma, cerebrospinal fluid, urine, blood, saliva, tears, lavage fluid, milk, water, cell / tissue culture supernatants, etc. The scope of the invention is not intended to be limited to any particular type or kind of sample either now known or later developed in the future.

By way of example, the at least one hollow element 42 (see Figures 2a, 3a, 3b) having the at least one narrow interior channel 44 (see Figures 2a, 3a, 3b) may be functionalized so as to form the reaction vessel generally indicated as 40 (see Figures 2a, 3a, 3b) in order to implement the present invention as disclosed in the aforementioned PCT applications, which are hereby incorporated by reference in their entirety. The scope of the invention is not intended to be limited to any particular type or kind of hollow element either disclosed therein, now known or later developed in the future, e.g., including the type of material, the external dimensions (length, diameter, width, etc.), the dimensions of the interior channel, etc.

By way of example, the concentration of the substantial enrichment and pre- concentration of the sample, including the at least one desired analyte of interest that may be further processed at a substantially higher concentration, is described to be in a range of about a 10-50 fold higher concentration; however, the scope of the invention is not intended to be strictly limited to any particular concentration or range of concentrations. Embodiments are envisioned with the spirit of the underlying invention to include other concentrations or ranges of concentrations depending, e.g., on the sample being further processed; depending, e.g., on the desired analyte of interest that may be further processed; or depending, e.g., on the low level antigen detections in relation to the sample. Moreover, the scope of the invention is intended to include other concentrations or other ranges of concentration depending on the sample being processed, the desired analyte deemed of interest, or the low level antigen being detected that is either now known or later developed in the future. By way of example, Figure 1 b shows steps 22, 24, 26 and 28 of a method generally indicated as 20 for the provisioning of the substantial enrichment and pre- concentration 18 of the sample 16 from the narrow interior channel 44, and the further processing of the substantial enrichment and pre-concentration of the sample, may include one or more of the following steps:

step 22 for releasing the at least one desired analyte of interest, including by a pH elution process;

step 24 for neutralizing the at least one desired analyte of interest in order for its rebinding downstream, including in a quantification section of an immunoassay instrument; and/or

step 26 for rebinding the at least one desired analyte of interest; and/or analysing the at least one desired analyte of interest, including in the analysis chamber of the immunoassay instrument, to detect the at least low level antigen in relation to the sample.

According to some embodiments of the present invention, and by way of example, the steps 22, 24, and 26 may be implemented by, or in conjunction with, a further processing device, apparatus or equipment 60 (see Figure 2b, 3a and 3b). Moreover, one skilled in the art would be able to configure the further processing device, apparatus or equipment 60 to implement the functionality of steps 22, 24, 26 without undue experimentation based at least partly on that disclosed herein.

Further, and by way of example, according to some embodiments of the present invention, the at least one desired analyte of interest may be released from the narrow interior channel 44 (Figures 2a, 3a, 3b), including by a pH elution process, to the further processing device, apparatus or equipment 60, where it is subsequently neutralized in step 24 and rebound in step 26. The scope of the invention is not intended to be limited to the manner, way or technique in which the at least one desired analyte of interest is released from the narrow interior channel 44, and is intended to includes other manners, ways or techniques for releasing the at least one desired analyte of interest from the narrow interior channel 44 either now known or later developed in the future. In addition to, or in lieu of, neutralizing and/or rebinding, the scope of the invention is intended to includes other manners, ways or techniques for further processing the at least one desired analyte of interest either now known or later developed in the future.

Once further processed, the further processing device, apparatus or equipment 60 (See Figures 3, 3a, 3b) provides the substantial enrichment and pre- concentration 18 as a further processed substantial enrichment and pre- concentration generally indicated by reference label 18a of the sample 16.

According to some embodiments of the present invention, the method may also include still further processing the further processed substantial enrichment and pre- concentration 18a with a step 30 for providing it to a subsequent reaction vessel generally indicated as 50 including at least one subsequent hollow element 52 having the at least one subsequent narrow interior channel 54 functionalized with the at least one subsequent enzyme, antibody or other affinity moiety selected to further concentrate the at least one desired analyte of interest present in the substantial enrichment and pre-concentration of the sample, e.g., as shown in Figures 2a, 3a, 3b. The at least one subsequent hollow element 52 is configured to receive the substantial enrichment and pre-concentration 18a of the sample 16, and provide a subsequent substantial enrichment and pre-concentration generally indicated as 18b of the sample 16 from the at least one subsequent narrow interior channel 54 so that the at least one desired analyte of interest may be further processed, if desired, in one or more of the subsequent further processing devices, apparatus or equipment 60a, 60b, 60c, 60d, 60e and/or subsequent reaction 50a, 50b, 50c, 50d. By way of example, and consistent with that described herein, the further processing devices, apparatus or equipment 60a, 60b, 60c, 60d, 60e may include a quantification section or an analysis chamber as well as other types or kind of further processing devices, apparatus or equipment either now known or later developed in the future.

Figure 1 e shows the present invention in the form of a method generally indicated as 100 according to some embodiments. The method 100 features a step 102 for processing a sample in at least one hollow element having at least one narrow interior channel functionalized with at least one enzyme, antibody or other affinity moiety selected in relation to a treatment of the sample; and a step 104 for providing a pre-treated sample from the at least one narrow interior channel that has either a substantial enrichment and pre-concentration of the sample, or a depletion of an interfering substance in the sample, or an activation of a latent molecule in the sample, or a degradation of the sample so to allow a subsequent capture of at least one desired analyte of interest, or a relevant molecule, or a latent molecule, or a reactive moiety in a downstream further processing of the pre-treated sample.

The treatment of the sample may include by way of example, sample enrichment, sample depletion, sample activation and sample degradation, consistent with that disclosed herein, which are provided by way of example. Moreover, the scope of the invention is not intended to be limited to any particular type or kind of treatment, and is intended to include other types or kinds of treatments of the sample either now known or later developed in the future. Figure 2

By way of example, Figure 2a shows the basic apparatus generally indicated as 40 in block diagram form, according to some embodiments the present invention, which the reaction vessel 40 consistent with that described herein.

By way of example, Figure 2b shows the further processing device, apparatus or equipment 60 for further processing the substantial enrichment and pre- concentration 18 of the sample 16, according to some embodiments of the present invention. Consistent with that disclosed herein, the further processing device, apparatus or equipment 60 may include a processing module 62 to implement the releasing functionality described herein, including by a pH elution process; a processing module 64 to implement the neutralizing functionality described herein; a processing module 66 to implement the rebinding functionality described herein; and a processing module 68 to implement the analysing functionality described herein, which may take the form of the analysis chamber of the immunoassay instrument. Moreover, the scope of the invention is not intended to be limited to any particular further processing of the substantial enrichment and pre-concentration 18 of the sample 16, and is intended to include other types or kinds of further processing functionality either now known or later developed in the future.

By way of example, Figure 2c shows apparatus generally indicated as 70 according to some embodiments of the present invention that includes the reaction vessel 40 in combination with the reaction vessel 50, as shown in Figure 3 for processing the sample 16, consistent with that disclosed herein. The apparatus 70 may also include the further processing device, apparatus or equipment shown in Figure 2b for processing and providing the substantial enrichment and pre- concentration 18 of the sample 16 from the reaction vessel 40 to the reaction vessel 50, consistent with that disclosed herein.

Figure 2d shows the present invention in the form of apparatus generally indicated as 1 10 according to some embodiments. The apparatus features at least one hollow element having at least one narrow interior channel functionalized with at least one enzyme, antibody or other affinity moiety selected in relation to a treatment of a sample, where the at least one hollow element is configured to:

receive the sample, and

provide a pre-treated sample from the at least one narrow interior channel that has either a substantial enrichment and pre-concentration of the sample, or a depletion of an interfering substance in the sample, or an activation of a latent molecule in the sample, or a degradation of the sample so to allow a subsequent capture of at least one desired analyte of interest, or a relevant molecule, or a latent molecule, or a reactive moiety in a downstream further processing of the pre-treated sample.

The present invention will now be described in further detail in relation to other specific example:

For example, the flow of materials or samples through cartridge(s) of the proprietary analyzer, developed and assigned to the assignee of the instant patent application, and disclosed in the aforementioned PCT applications, also lends itself to a unique feature of an analytical instrument, namely that of an in-line processing of the sample for improved assay outcome. This would entail an on-line selection or de-selection of the target analyte to isolate its reactive element or component within a "purification" Glass NanoReactor (GNR), such as reaction vessel 40 in Figures 3a and 3b, followed by its analysis (e.g., within a "capture" GNR) in one or more subsequent reaction vessels 50, 50a, 50b, 50c, 50d. (The GNR is also

interchangeably referred to herein as the hollow element that is functionalized with an enzyme, antibody or other affinity moiety so as to form the so-called reaction vessel.) As an example, an analyte could be treated to a series of enzymes E1 , E2, E... (immobilized within different "purification" GNR's) to isolate bio-molecules of interest, that would be subsequently captured and/or characterized for different antigens or factors via appropriately coated capture GNRs.

The concept of the invention may be visualized and include the following process flow: l_E1_|→ |_E2_|→ |_E..._|→ |_Ab1_|→ |_AB2_|→ |_Ab..._|, where each figure of | | represents one or more hollow element or GNR immobilized in a channel of a cartridge. The letters E denote an "immobilized component," as identified in Figures 3a, 3b, which is able, e.g., to convert an analyte of interest to another compound which is more readily measured. In Figures 3a, 3b, the numbers 1 through X denote one or more GNRs that may have either identical or different immobilized content. The ellipsis marks indicates that the numbers can continue to a high value (plex). The arrows indicate the flow of the sample as it is processed and further processed.

Examples of E components may include enzymes and other affinity moieties that process the analyte of interest into a more readily measured compound.

In a specific embodiment, described by way of example, the reaction vessels 40, 50 50a (functionalized with enzyme or immobilization components E1 , E2 and E3) could be protease specific "purification" GNR's. In particular, the reaction vessel 40 (aka functionalized hollow element (GNR) E1 ) could be an MMP-2 (matrix metalloproteinase 2) embedded GNR', the reaction vessel 50 (aka functionalized hollow element (GNR) E2) could be an MMP-9 (matrix metalloproteinase 9) embedded GNR, and the reaction vessel 50a (aka functionalized hollow element (GNR) E...) could be a thrombospondin (TSP-1 ) embedded GNR. The reaction vessels 50b, 50b 50d (aka functionalized hollow elements (GNRs) Ab1 , 2 and 3) will be capture GNR's coated with antibodies specific to TGF-B. Upon sample

introduction, platelets will be acted upon by the proteases in functionalized hollow elements E1 , 2 and 3 to release TGF-B that would be subsequently captured by the reaction vessels 50b, 50b 50d (aka functionalized hollow elements (GNRs) Ab1 , 2 and 3).

According to some embodiments, the present invention may also take the form of an alternative configuration that may include the following process flow: l_E1_|→ |_Ab1_|→ |_E2_|→ |_AB2_|→ |_E..._|→ |_Ab..._|, where the enzyme and antibody based GNRs are alternated for processing and further processing the sample and the substantial enrichment and pre-concentration of the sample.

In addition, an alternate use of these sequential GNRs is the removal of interfering substances. It is well known in the industry that certain components of a sample may adversely affect the ability of the analyte to give a true response. These compounds are generally called interfering substances and only a couple of solutions exist for the processing of such samples. The simplest is for the method to detect and remove the interfering substances. The alternative is a pre-treatment of sample with the intent of removing the interfering substance. With the invention set forth herein, the interfering substance can be rendered harmless to the analysis by the affinity removal or other type of conversion or removal of the interfering

substance.

The Microfluidic Technology

By way of example, the term "microfluidics" is generally understood to mean or deal with the behavior, precise control and manipulation of fluids that are geometrically constrained to a small, typically sub-millimeter, scale. In the present application, the microfluidic technology described herein is intended to include technology dimensioned in a range of about 20 micron to about 1000 microns, although the scope of the invention is not intended to be limited to any particular range. By way of example, according to some embodiments the reaction vessels 40, 50 etc. may be characterized by the at least one narrow interior channel of the at least one hollow element having one or one of the following features: The at least one narrow interior channel of the at least one hollow element may be configured or designed with a cavity or chamber having a very small inside diameter that is approximately 10μηη inner diameter (ID)) and a length-to-I.D. aspect ratio of approximately 20:1 , which is approximately 200μηη L; and the at least one hollow element or the at least one subsequent hollow element may be configured or designed as a honeycomb structure having a multiplicity of cavities or chambers having a very small inside diameter that is approximately 10μιτι inner diameter (ID)), both consistent with that disclosed in the aforementioned PCT applications.

However, the scope of the invention is intended to include using reaction vessels having other dimensional configurations either now known or later developed in the future. For example, as fiber optic drawing technology evolves and optical fiber having the structural integrity to implement the present invention is developed in the future having, e.g., smaller dimensional characteristics, the scope of the invention is intended to include such future developments, particularly as it relates to both the dimension of the narrow interior channel, as well as exterior dimensionality of the over reaction vessel.

Figures 4a to 4c: Sample Enrichment Precolumn for immunoassay device Figures 4a to 4c show other aspects of the present invention according to some embodiments thereof, which can provide a method or technique to enrich analytes of interest from within a blood sample for subsequent immunoassay analysis. The invention according to these embodiments provides another method or technique for the automated removal of cells and platelets from the whole blood sample, and a significant enrichment of sample providing 10 - 50 times greater antigen to enable detection of trace analytes.

The method uses restricted access beads or hollow elements within a precolumn (Figure 4a) to accomplish two distinct actions both which serve to enrich the target analytes and improve the performance of an immunoassay. First, the method removes blood cells from a sample thus enabling whole blood to be presented to an instrument as opposed to plasma or serum. The removal of blood cells uses a gel filtration approach (Figure 4b) where blood cells and platelets are excluded from narrow interior channels of beads used in the pre-column.

Conversely, the soluble plasma component of the blood sample which contains the analytes of interest can enter the interior channels of the beads albeit at a slower rate (Figure 4c). As such, the soluble plasma component of the sample is delayed thus allowing the blood cells to elute ahead of the soluble plasma component of interest and effectively removing the cells from the downstream analysis.

The Scope of the Invention

Embodiments shown and described in detail herein are provided by way of example only; and the scope of the invention is not intended to be limited to the particular configurations, dimensionalities, and/or design details of these parts or elements included herein. In other words, a person skilled in the art would

appreciate that design changes to these embodiments may be made and such that the resulting embodiments would be different than the embodiments disclosed herein, but would still be within the overall spirit of the present invention.

It should be understood that, unless stated otherwise herein, any of the features, characteristics, alternatives or modifications described regarding a particular embodiment herein may also be applied, used, or incorporated with any other embodiment described herein. Also, the drawing herein are not drawn to scale.

Although the invention has been described and illustrated with respect to exemplary embodiments thereof, the foregoing and various other additions and omissions may be made therein and thereto without departing from the spirit and scope of the present invention.

Claims

WHAT WE CLAIM IS:

1 . A method comprising:

processing a sample in at least one hollow element having at least one narrow interior channel functionalized with at least one enzyme, antibody or other affinity moiety selected in relation to a treatment of the sample; and

providing a pre-treated sample from the at least one narrow interior channel that has either a substantial enrichment and pre-concentration of the sample, or a depletion of an interfering substance in the sample, or an activation of a latent molecule in the sample, or a degradation of the sample so to allow a subsequent capture of at least one desired analyte of interest, or a relevant molecule, or a latent molecule, or a reactive moiety in a downstream further processing of the pre-treated sample.

2. A method according to claim 1 , wherein the at least one narrow interior channel is functionalized with an immobilized affinity molecule capable of binding the sample so as to extract the sample, including a bacterium sub-class, from large sample volumes.

3. A method according to claim 2, wherein the method comprises subjecting the at least one hollow element to a physico-chemical elution, including a change in pH or ionic strength, that would enable the release an isolated analyte that has been concentrated to allow its capture and subsequent recognition downstream by at least one subsequent hollow element having at least one subsequent narrow interior channel functionalized with at least one subsequent enzyme, antibody or other affinity moiety.

4. A method according to claim 1 , wherein the at least one narrow interior channel is functionalized with an immobilized protein or a factor capable of removing an interfering substance so as to allow a downstream recognition of the relevant molecule by at least one subsequent hollow element having at least one subsequent narrow interior channel functionalized with at least one subsequent enzyme, antibody or other affinity moiety.

5. A method according to claim 1 , wherein the at least one narrow interior channel is functionalized with an immobilized protein or a factor capable of activating a latent molecule so as to allow its recognition and downstream capture by at least one subsequent hollow element having at least one subsequent narrow interior channel functionalized with at least one subsequent enzyme, antibody or other affinity moiety.

6. A method according to claim 1 , wherein the at least one narrow interior channel is functionalized with an immobilized enzyme capable of degrading the sample so as to allow access to the reactive moiety which would be recognized and captured downstream by at least one subsequent hollow element having at least one subsequent narrow interior channel functionalized with at least one subsequent enzyme, antibody or other affinity moiety.

7. Apparatus, including a reaction vessel, comprising:

at least one hollow element having at least one narrow interior channel functionalized with at one least one enzyme, antibody or other affinity moiety selected in relation to a treatment of a sample;

the at least one hollow element configured to:

receive the sample, and

8. Apparatus according to claim 7, wherein the at least one narrow interior channel is functionalized with an immobilized affinity molecule capable of binding the sample so as to extract the sample, including a bacterium sub-class, from large sample volumes.

9. Apparatus according to claim 8, wherein the method comprises subjecting the at least one hollow element to a physico-chemical elution, including a change in pH or ionic strength, that would enable the release an isolated analyte that has been concentrated to allow its capture and subsequent recognition downstream by at least one subsequent hollow element having at least one subsequent narrow interior channel functionalized with at least one subsequent enzyme, antibody or other affinity moiety.

10. Apparatus according to claim 7, wherein the at least one narrow interior channel is functionalized with an immobilized protein or a factor capable of removing an interfering substance so as to allow a downstream recognition of the relevant molecule by at least one subsequent hollow element having at least one subsequent narrow interior channel functionalized with at least one subsequent enzyme, antibody or other affinity moiety.

1 1 . Apparatus according to claim 7, wherein the at least one narrow interior channel is functionalized with an immobilized protein or a factor capable of activating a latent molecule so as to allow its recognition and downstream capture by at least one subsequent hollow element having at least one subsequent narrow interior channel functionalized with at least one subsequent enzyme, antibody or other affinity moiety.

12. Apparatus according to claim 7, wherein the at least one narrow interior channel is functionalized with an immobilized enzyme capable of degrading the sample so as to allow access to the reactive moiety which would be recognized and captured downstream by at least one subsequent hollow element having at least one subsequent narrow interior channel functionalized with at least one subsequent enzyme, antibody or other affinity moiety.

13. A method comprising:

14. A method according to claim 13, wherein the at least one enzyme, antibody or other affinity moiety is selected to retain the at least one desired analyte of interest in the at least one narrow interior channel.

15. A method according to claim 14, wherein the method comprises releasing the at least one desired analyte of interest, including by a pH elution process.

16. A method according to claim 15, wherein the method comprises neutralizing the at least one desired analyte of interest in order for its rebinding downstream, including in a quantification section of an immunoassay instrument.

17. A method according to claim 16, wherein the method comprises rebinding the at least one desired analyte of interest.

18. A method according to claim 17, wherein the method comprises analysing the at least one desired analyte of interest, including in an analysis chamber of an immunoassay instrument, to detect the at least low level antigen in relation to the sample.

19. A method according to claim 13, wherein the sample is a biological or patient sample, including one or more of the following: serum, plasma, cerebrospinal fluid, urine, blood, saliva, tears, lavage fluid, milk, water, cell / tissue culture supernatants.

20. A method according to claim 13, wherein the method further comprises providing the substantial enrichment and pre-concentration of the sample to at least one subsequent hollow element having at least one subsequent narrow interior channel functionalized with at least one subsequent enzyme, antibody or other affinity moiety selected to further concentrate the at least one desired analyte of interest present in the substantial enrichment and pre-concentration of the sample.

21 . A method according to claim 20, wherein the method comprises arranging the at least one hollow element and the at least one subsequent hollow element for in-line processing.

22. A method according to claim 21 , wherein the in-line processing includes an on-line selection or de-selection of the at least one analyte of interest to isolate its reactive element or component, including by treating the at least one analyte of interest to a series of enzymes to isolate bio-molecules of interest that may be subsequently captured or characterized for different antigens or factors using a series of hollow elements having appropriately functionalized narrow interior channels with appropriate enzymes, antibodies or other affinity moieties.

23. A method according to claim 13, wherein the at least one enzyme, antibody or other affinity moiety releases the at least one desired analyte of interest from the sample.

24. A method according to claim 13, wherein the at least one enzyme, antibody or other affinity moiety is an immobilized component that convert the at least one analyte of interest to another compound which is more easily measured.

25. A method according to claim 13, wherein the at least one enzyme, antibody or other affinity moiety comprises some combination of identical or different enzymes, antibodies or other affinity moieties.

26. A method according to claim 13, wherein the at least one enzyme, antibody or other affinity moiety removes or converts an interfering substance in the sample that would otherwise adversely affects the ability of the at least one analyte of interest to provide a true response when treated.

27. A method according to claim 13, wherein the at least one enzyme, antibody or other affinity moiety comprises a combination of identical or different enzymes, antibodies or other affinity moieties.

28. A method according to claim 13, wherein the at least one narrow interior channel of the at least one hollow element is configured or designed with a cavity or chamber having a very small inside diameter that is approximately 10μηη inner diameter (ID) and a length-to-I.D aspect ratio of approximately 20:1 , which is approximately 20Όμηη L.

29. A method according to claim 13, wherein the at least one hollow element or the at least one subsequent hollow element is configured or designed as a honeycomb structure having a multiplicity of cavities or chambers having a very small inside diameter that is approximately 10μηη inner diameter (ID).

30. Apparatus, including a reaction vessel, comprising:

the at least one hollow element configured to:

receive the sample, and

provide a substantial enrichment and pre-concentration of the sample from the at least one narrow interior channel so that the at least one desired analyte of interest may be further processed at a substantially higher concentration, including in a range of about a 10-50 fold higher concentration, thus enabling at least low level antigen detections in relation to the sample.

31 . Apparatus according to claim 30, wherein the at least one enzyme, antibody or other affinity moiety is selected to retain the at least one desired analyte of interest in the at least one narrow interior channel.

32. Apparatus according to claim 31 , wherein the apparatus comprises a further processing device, apparatus or equipment configured to released the at least one desired analyte of interest, including by a pH elution process.

33. Apparatus according to claim 32, wherein the further processing device, apparatus or equipment is configured to neutralize the at least one desired analyte of interest in order for its rebinding downstream, including in a quantification section of an immunoassay instrument.

34. Apparatus according to claim 33, wherein the further processing device, apparatus or equipment configured to rebind the at least one desired analyte of interest.

35. Apparatus according to claim 34, wherein the further processing device, apparatus or equipment is configured to analyze the at least one desired analyte of interest, including in an analysis chamber of an immunoassay instrument, to detect the at least low level antigen in relation to the sample.

36. Apparatus according to claim 30, wherein the sample is a biological or patient sample, including one or more of the following: serum, plasma, cerebrospinal fluid, urine, blood, saliva, tears, lavage fluid, milk, water, cell / tissue culture supernatants.

37. Apparatus according to claim 30, wherein the apparatus further comprises at least one subsequent hollow element having at least one subsequent narrow interior channel functionalized with at least one subsequent enzyme, antibody or other affinity moiety selected to further concentrate the at least one desired analyte of interest present in the substantial enrichment and pre-concentration of the sample, and being configured to receive the substantial enrichment and pre-concentration of the sample.

38. Apparatus according to claim 37, wherein the at least one hollow element and the at least one subsequent hollow element are configured for in-line

processing.

39. Apparatus according to claim 38, wherein the in-line processing includes an on-line selection or de-selection of the at least one analyte of interest to isolate its reactive element or component, including by treating the at least one analyte of interest to a series of enzymes to isolate bio-molecules of interest that may be subsequently captured or characterized for different antigens or factors using a series of hollow elements having appropriately functionalized narrow interior channels with appropriate enzymes, antibodies or other affinity moieties.

40. Apparatus according to claim 30, wherein the at least one enzyme, antibody or other affinity moiety releases the at least one desired analyte of interest from the sample.

41 . Apparatus according to claim 30, wherein the at least one enzyme, antibody or other affinity moiety is an immobilized component that convert the at least one analyte of interest to another compound which is more easily measured.

42. Apparatus according to claim 30, wherein the at least one enzyme, antibody or other affinity moiety comprises some combination of identical or different enzymes, antibodies or other affinity moieties.

43. Apparatus according to claim 30, wherein the at least one enzyme, antibody or other affinity moiety removes or converts an interfering substance in the sample that would otherwise adversely affects the ability of the at least one analyte of interest to provide a true response when treated.

44. Apparatus according to claim 30, wherein the at least one enzyme, antibody or other affinity moiety comprises a combination of identical or different enzymes, antibodies or other affinity moieties.

45. Apparatus according to claim 30, wherein the at least one enzyme, antibody or other affinity moiety releases the analyte of interest from the sample so as to form a concentrated analyte of interested that is more readily measured.

46. Apparatus according to claim 30, wherein the at least one enzyme, antibody or other affinity moiety removes or converts an interfering substance in the sample that would otherwise adversely affects the ability of the analyte of interest to provide a true response when treated.

47. Apparatus according to claim 30, wherein the at least one narrow interior channel of the at least one hollow element is configured or designed with a cavity or chamber having a very small inside diameter that is approximately 10μηη inner diameter (ID) and a length-to-I.D aspect ratio of approximately 20:1 , which is approximately 200μηη L.

48. Apparatus according to claim 30, wherein the at least one hollow element or the at least one subsequent hollow element is configured or designed as a honeycomb structure having a multiplicity of cavities or chambers having a very small inside diameter that is approximately 10μηη inner diameter (ID).