JP2003520961A - Methods and devices for separating and detecting nucleic acids - Google Patents

Abstract

(57) SUMMARY Thin-layer gels for separating and detecting target molecules (eg, analytes) and methods for their use are described. The gel is optionally strengthened to facilitate removal from the electrophoresis device. Here, the gel matrix comprises an immobilized (eg, covalently bound) capture probe that can specifically hybridize or bind to the analyte to be detected. The gel is thin and very short assay times are achieved, which facilitates rapid detection of the analyte. Assay devices are also described that are suitable for use with an electrophoretic assay system for the detection, or separation and detection of an analyte in a biological sample.

Description

Detailed Description of the Invention

RELATED APPLICATION This application is filed on January 19, 2000, US Provisional Application No. 60 / 176,8
No. 39 (the entire teachings of which are incorporated herein by reference).

BACKGROUND OF THE INVENTION Separation and detection of nucleic acid molecules and fragments is desirable for diagnostic, analytical, and research purposes. Nucleic acid includes DNA, RNA, and analogs thereof.
Different species of microorganisms (eg, bacteria, viruses, fungi, and / or parasites) have unique gene (DNA and / or RNA) sequences that detect the presence or absence of a particular microorganism. Can be used for. The consensus sequence of nucleic acids is also recognized within a population of microorganisms (eg, in bacteria), which can be used to detect the presence or absence of members of this particular population. Furthermore, DN
Disease or dysfunction can occur if A or RNA is modified, eg, by a single nucleotide polymorphism (SNP) or mutation. Detection of polymorphisms and mutations may facilitate early treatment during the life of a patient, in the form of medical care or education, to improve quality of life. For example, if genetic analysis indicates that the child is born with cystic fibrosis, an early medical treatment that may minimize the complications of the disease may be provided.

A biological sample can consist of a heterogeneous mixture of components such as proteins and nucleic acids. The ability to rapidly separate components and identify a target molecule (eg, a particular DNA, RNA or protein) in a sample, especially for diagnostic applications, is a limiting factor in developing useful diagnostic tests. obtain. For example, if a patient requires an infusion of platelets, knowledge of whether the platelets are contaminated with bacteria at the time of transfusion is required. During the time required to complete the test, the bacteria will
Continue to increase. Thus, the usefulness of platelets may change over time.

Electrophoresis on a gel is one method traditionally used to separate complex samples containing nucleic acid molecules and fragments into individual bands with unique sequences. Both the weight and charge of a molecule or fragment can affect the rate of its migration through the electrophoretic medium. For nucleic acids with uniform charge to mass ratio,
The larger the nucleic acid, the slower its migration within the gel. The distance the sample must travel before it is separated into its constituent parts also affects the speed of separation using this method. Traditional methods of analyzing nucleic acids typically require highly trained technicians and take 2-48 hours to complete, depending on the sample and type of equipment used.

Clearly, there is a need for methods and devices for the rapid and accurate separation and detection of target molecules in biological samples.

SUMMARY OF THE INVENTION The present invention detects, separates or separates components or target molecules (eg, analytes) in a biological sample by moving the components into the vicinity of a capture probe by an electric field.
A system for purification and / or enrichment, wherein the component specifically binds to or hybridizes to a capture probe.
The system includes a housing (eg, an electrophoretic cassette to house at least two electrodes); one or more structures to house a thin gel matrix with capture probes; an ion supply to support an electric field. A source (eg, a relaxed salt solution) can be provided. The capture probe can optionally be contained within a gel matrix such as agarose. If necessary, this device
Structures for holding the lamella gel or for holding the reinforced lamella gel; structures may be provided for introducing the sample into the sample compartment within the system, where the sample is potentially , A component that binds to or hybridizes to the capture probe (eg, a nucleic acid sequence that is substantially complementary to the capture probe). The system may further include a power supply, or may include structure for connecting the system to the power supply, or for connecting parts of the system to the power supply.

This system of the invention can be used to determine the presence or absence of a component or target molecule. For example, the sample can be transferred to an electrophoresis cassette (
For example, the sample is introduced into the sample compartment of the cassette and an electrical force (eg, a voltage gradient) is applied across the electrodes of the cassette such that one or more components of the sample are diluted from the sample compartment. Through the layer gel matrix). One or more capture probes are bound to this gel.
As used herein, a capture probe is bound to the gel by loading, immobilizing, trapping, and / or chemically ionically and covalently binding the capture probe. Can be bound to the gel.
This capture probe may be referred to herein as a ligand. For example, if the target molecule (eg, analyte) to be detected is a nucleic acid having a nucleotide sequence that is substantially complementary to the nucleotide sequence of the capture probe, then the nucleic acid is
Nucleic acid molecules retained within the lamella gel, while lacking complementary sequences, migrate through the gel. This system of the invention, when used in a method of detection, assay, or analysis, binds a target molecule to a capture probe by virtue of one or more chemical or physical properties exhibited by the capture probe-target complex. This property can be detected and is known to those skilled in the art. In one embodiment, the capture probe-target complex comprises a detectable label.

In one embodiment, the system for capturing an analyte comprises an electrophoresis cassette. The electrophoresis cassette includes a housing (eg, a base having a set of electrode channels), a barrier inserted between the electrode channels, a barrier having at least one migration channel extending between the electrode channels, and a bounded 1
A set of expansion slots and openings to the migration channels are provided. The first electrode extends into the first electrode channel and the second electrode extends into the second electrode channel. A capture gel holder (gel capture holder) can be housed in the expansion slot. The capture gel holder (gel capture holder) may have openings aligned with the migration channels. The thin gel is positioned in the opening of the capture gel holder. In one embodiment, the capture gel holder is a comb. The lamellar gel has a matrix that can be a polymer mesh; optionally, the matrix is
It can be non-conductive.

In one embodiment, the cassette comprises an evaporation cover positioned to overlie the base of the cassette. The evaporation cover has at least one opening for the capture gel holder, at least one opening for venting gas, and / or an opening to allow the sample well forming comb to enter the migration channel. obtain. This cassette is for housing the teeth of the capture gel holder,
A set of wash wells may be provided. These electrodes may have a set of terminals extending through the evaporation cover. These terminals can be flush with the top of the evaporation cover.

In one embodiment, the capture gel holder has a handle and a plurality of teeth protruding from the handle. One or more of the teeth can have holes through the teeth. The polymeric material (eg, a non-conductive material, for example) may occupy an empty ring inside the hole of the tooth and thus be stretched over or into the hole of the tooth. This material may support a gel, such as a gel matrix. The hole may be bounded by a recessed central region, preferably surrounded by this region,
And the recessed central region and the flange may be bounded by, or surrounded by, a flange over the hole to facilitate the release of gas. At least one tooth has a shape (eg, a keyed shape) adapted to fit the cassette in a particular or special orientation.

In one embodiment, the thin layer gel is used to separate and detect target molecules (eg, analytes), eg, nucleic acid sequences, nucleic acid analogs, wherein such analogs are used in a sample. For example, a peptide nucleic acid (PNA), a modified nucleic acid sequence, or a peptide having a particular binding affinity, a particular structure, or a particular amino acid sequence. More specifically, the present invention provides a polymeric gel matrix, optionally enhanced to allow removal from a device (eg, an electrophoretic cassette), wherein the polymeric gel matrix is Contains a capture probe that can specifically hybridize or bind to a target molecule. As described herein, this gel is a "thin layer". The term "thin layer" as used herein refers to a thick mass of gel matrix. The gel matrix of the present invention has the properties of either a filter or a sieve, so that it retains one or more sample components in the gel matrix while allowing other components to pass through. The gel matrix of the present invention is operable so that it can be placed in, for example, an electrophoresis cassette and removed. By reducing the thickness of the gel matrix more, assay rates can be achieved. However, the term "thin layer" also refers to the characteristics of selective permeability and ease of handling so as to reduce the amount of time required to achieve the desired detection, separation, or concentration. You can Importantly, using the methods and apparatus described herein, no pre-amplification of sample detection is required, yet the sensitivity of this rapid assay is more complex than analytes. Comparable to the time-consuming way.

The gel can include any material that can be used in an electrophoretic assay system to detect, separate, or concentrate an analyte, eg, in the form of polyacrylamide, agarose, starch, or dextran. The gel may be formulated so that it is stable enough to be repositioned, removed, or physically moved by manual or mechanical means. The gel matrix may be provided with a reinforcement that acts to reinforce the gel, thereby facilitating handling. When this lamellar gel matrix is cast on a reinforcing material, this lamellar gel matrix is called a reinforced lamellar gel. In one embodiment, the lamina gel reinforcement includes a non-conductive porous material (eg, mesh, web, mat, or felt). In the second embodiment,
The lamellar gel contains polymeric additives, crosslinkers, or other chemical agents, which may be incorporated into the gel, thus structurally strengthening the gel matrix. The reinforcement increases the strength and durability of the gel matrix, which makes it tear or stretch resistant. In addition, the gel can be used with a holder that facilitates migration and processing of the gel within a detector or detection station for detecting binding of target molecules to cassette probes retained within the gel.

In another embodiment, a highly sensitive, non-amplifying, rapid assay for the detection of target molecules in a biological sample is provided. Target molecules, such as samples (eg, samples suspected of being contaminated with unwanted species), eg, microbial species (eg, bacteria, fungi, or viruses), are treated as described herein. To utilize that nucleic acids characterized as present in this species, preferably known to be unique to that species, can hybridize to the capture probe bound to the gel matrix of the capture gel holder. to enable. This hybridized nucleic acid can thereby be captured on a gel and then detected and reported.

In this embodiment of the invention, the biological sample is first diluted with a suitable diluent and then centrifuged at high speed to pellet any target bacteria present in the sample. The pelleted bacteria are treated in a manner that solubilizes the membrane components and renders the target bacteria suitable for hybridization with a particular nucleic acid probe. A buffer containing alkaline phosphatase-conjugated reporter probe is added to the sample and the sample is heated to a temperature sufficient for denaturation of the target nucleic acid and hybridization of the reporter probe. The samples are cooled, and an aliquot of each sample is loaded into a pre-warmed electrophoresis cassette as described herein and voltage is applied. These samples pass through a capture comb, which is also referred to herein as a capture gel holder, containing capture probes specific for the target bacterial nucleic acids. This capture probe is immobilized on a thin layer gel. The comb is then dip washed and moved to a slot in the cassette, which is upstream of the original electrophoretic pathway. Again, the voltage is briefly applied to electrophoretically wash the comb. The comb is placed in conditioning buffer for a period of time, then removed and placed flat on the reader tray. The enzyme substrate is added in an amount sufficient to react with the reporter probe that is hybridized to the target immobilized on the gel. This reader tray is then scanned by the radar,
Then any signal present is detected and reported. Detection of the signal indicates the presence of the target bacterium in the sample.

In another embodiment, for example, a sample suspected of bacterial contamination may be cultured to grow any colonies of bacteria present in the sample, and the culture is agarose gel encapsulated. Transfer to a thin gel matrix by blotting the sample with a polyester or nylon membrane. The transcribed bacterial colonies can be exposed to conditions that result in lysis of the bacteria and release of the analyte (s) (eg, the nucleic acids contained therein). The nylon membrane can then be sandwiched against a thin gel matrix containing a capture probe (specific for the bacterium to be detected) and any air bubbles between the two removed (eg,
By gentle pressure). The sandwich can then be placed in an electrophoretic device, and under appropriate conditions an electric current is applied, and the bacterial component containing the analyte (s) to be detected is electrophoresed (ie, Move) to contact the immobilized capture probe. If the bacterial analyte to be detected is present in the sample, it will hybridize or bind to the immobilized probe and remain immobilized in the gel (ie, not migrate further). )
. Bound analyte can then be detected, for example, by hybridization of the analyte to a reporter probe, and reported as described herein.

Further, the methods and devices described herein allow for rapid culture and purification of hybridized nucleic acids. For example, using the methods and devices described herein, the target nucleic acid can be rapidly isolated from the complex sample and specifically by the immobilized capture probe of the gel on the capture comb. To be captured. Once the target nucleic acid has been captured and the remaining contaminants from the sample have been removed from the presence of the target, the capture combs have been loaded into different sets of slots, separate vessels, or fixed in the same electrophoresis cassette. The target / probe complex may be removed by another device subject to conditions that release the target from the probe, thus the target molecule is recovered from the sample and used in further procedures such as the polymerase chain reaction. obtain.

In addition, confirmatory tests for the presence of particular polymerase chain reaction (PCR) products can be performed using the methods and devices of the invention. The complex PCR reaction mixture can be contacted with the immobilized capture probe on the capture comb, and the specific PCR
The product can be captured and detected to confirm the correct performance of the PCR reaction.

As a result of the invention described herein, methods and devices are available for rapid assay and isolation of target molecules from complex biological samples. The invention described herein eliminates the need for complex and time consuming chemical reactions, and of background interference of the sample, as well as of environmental conditions that can be detrimental to accurate diagnostic and purification results. Substantially reduce or eliminate nucleic acid contamination. For example, using a method and apparatus as described herein, a wide panel of clinically relevant bacterial contaminations at levels of about 10 ⁴ to 10 ⁵ CFU / ml can lead to sample nucleic acid It can be detected without the need for PCR amplification. Accordingly, rapid assay formats are available herein for detecting bacterial contamination of biological products (eg, bacterial contamination of blood products such as platelets) using simplified sample processing.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the discovery that detection of target molecules can be readily accomplished by the use of a thin gel matrix containing immobilized capture probes. The capture and subsequent detection of the target molecule depends on the density of capture probes in the gel rather than the distance traveled by the target molecule through the thin gel matrix. Moreover, the incorporation of the reinforcement into the thin gel matrix facilitates its handling and its removal from the device, thereby enabling improved procedures for background signal removal and detection. This reinforcement may be provided by a separate material such as a non-conductive mesh or fibrous mat. Alternatively, the reinforcement may be a molecular reinforcement (eg, cross-linking molecule) incorporated within the lamellar gel matrix, which strengthens and / or reinforces the matrix.

In one particular embodiment, the invention provides a thin gel, an electrophoretic system for use with the thin gel, and a method of using the electrophoretic system with the thin gel. including. More specifically, the present invention relates to a reinforced thin layer gel,
Electrophoresis device or device, sample comb or capture gel holder for use with the electrophoretic device, and electrophoretic device and capture gel for separating and subsequently detecting an analyte from the sample Includes methods using holders (sample combs).

An electrophoresis cassette 190 according to the present invention is shown in FIGS. The cassette 190 has a base 192 and an evaporation cover 194 shown in FIG.

Referring to FIGS. 1 and 3, the base 192 of this electrophoresis cassette 190 is
It has a pair of electrode channels 92 and 94. In each of the electrode channels 92 and 94, there are a plurality of ribs 198 that cut each of these channels into sections.
Each rib 198 has a slot 200 through which an electrode 202 (eg, as shown in FIG. 3) extends. The slot 200 of this rib 198 is used to ensure that the electrode 202 is properly positioned as described in detail below. The electrode 202 extends the length of each of the electrode channels 92 and 94. In one embodiment, each of the electrodes 202 includes a pair of posts 204 and a strip of stainless steel or platinum 206 extending between the posts 204, as shown in FIG. A portion of the post 204 is shown in FIG. 3 for illustrative purposes, where the post 204 is not installed on the base 192 until after the evaporation cover 194 is installed. Each post 204 has a cylindrical rod 208, shown in cross section in FIG. 3, which is received in a hole 210 in the base 192, and a complete annular disk or head 212 as shown in FIG. Located on top of the evaporation cover 194. The post 204 is formed of stainless steel in one embodiment.

This base 192 comprises a plurality of channels, migration channels 9 as shown in FIG.
6 which extends between the electrode channels 92 and 94. Each migration channel 9
Within 6, there are a pair of enlarged slots 98 adapted to receive the teeth 218 of the capture gel holder 220. This capture gel holder 220 is further described below with reference to Figures 5-7G. An alternative embodiment of capture gel holder 66 is briefly described below. Whether the capture gel holder 66 can be used with a particular electrophoresis cassette 190 depends on a number of factors, including tooth spacing and size, and any keying features.

Referring to FIGS. 1-3, the base 192 of the electrophoresis cassette 190 has a pair of wash well sets 222 and 224 of FIG. Wash wells 22 for each set
2 and 224 are associated with migration channel 96 and have similarly spaced open wash wells 226. Each wash well 226 is adapted to receive one of the teeth 218 of the capture gel holder. Set of wash wells 222 and 224
Each wash well 226 of each has a rib or support 228 with a slot 230 for receiving the tooth 70 or 218 of the capture gel holder 60 or 220. The ribs 228 with slots 230 diverge, as shown in FIG. 5, so that they do not interfere with the capture gel 40 and are not aligned with the running channels 96 that have the capture gel holder 220 aligned and upright. The holes 76 in each tooth 218 having a tooth ensure that they are accessible by the solution in the wash well.

In one embodiment, the wash wells 226 of the set of wash wells 224 are
It has a larger volume or volume than another set of wash wells 222. The purpose of the difference in size is to allow the capture gel 40 to react with different amounts and concentrations of solutions or reactants. For example, in one embodiment, the set of wash wells 224 is referred to as the set of conditioner wells and another set 222 of wash wells is referred to as the set of wash wells.

Referring to FIGS. 2, 4A, and 4B, evaporation cover 194 has a plurality of vents or slots 234 for covering electrodes 202. The evaporation cover 194 further has a plurality of openings 236, which are generally square. This square opening 236 is provided with a plurality of teeth 382 of the sample well forming comb 380, as shown in FIG. 8A.
Can be passed to the migration channel 96.

Still referring to FIGS. 2, 4A, and 4B, the evaporation cover 194 has a plurality of slots 238 for receiving the teeth 218 of the capture gel holder 220, and as shown in FIG. Expansion slot 9 of 192 migration channel 96
Lying on eight. In one embodiment, this slot 238 has a rounded side edge 240 and a square side edge 242, as best shown in FIG. 4B. This slot 238 is formed so that the capture gel holder 220 can only pass through the slot 238 in a particular orientation, as described in more detail below with reference to FIGS. 5-7G. In one embodiment, the evaporation cover 194 is attached to the base 192 by ultrasonic welding.

The evaporation cover 194 does not cover the sets of wash wells 222 and 224. As shown above, the annular disc 212 of the post 204 is the electrode 202,
Lying on the evaporation cover 194. Evaporation cover 194 has a hole 246 through which the annular rod of post 204 extends.

Referring still to FIGS. 4A and 4B, the evaporation cover 194 is complementary to the base (192) of the electrophoresis cassette 190 as shown in FIGS.
a plurality of tabs 248 that are received in the notches 250. One of these tabs of tabs 248 (tab 252) is located slightly inside the position of the other tab 248, as shown in FIGS. 4A and 4B, so that this evaporation cover 194 is It may only be arranged on the base 192 in one direction.
In one embodiment, both the evaporation cover 194 and the base 192 have teeth 2
38 and the catches having "A", "B", "C" and "D" letter-like marks 256 adjacent to the slots for sets of wash wells and wash wells 222 and 224 and inserted into their respective components. The order (ie, steps) for receiving the teeth 218 of the gel holder 220 is shown. This step is described in further detail below.

Referring back to FIGS. 2 and 3, the electrophoresis cassette 190 includes a handle portion 258 on the base 192 to assist in the movement of the electrophoresis cassette 190.
Have.

The electrophoresis cassette 190 is used in connection with a capture gel holder in the method of capturing target molecules. The capture gel holder 220 as illustrated in FIG.
It has a plurality of teeth 218 and a handle portion 260. The handle portion 260 includes a groove 262 and a second groove or marker receiving portion 264 to aid in gripping.
Have.

Referring to FIGS. 5 and 6A, each tooth 218 has a thin central region 266 in which is located a hole 268 extending through tooth 218. Thinning this central region 266 prevents it from trapping air bubbles on the surface of the capture gel 40 and teeth 218 when the capture gel holder is inserted into slots 98 and 238 of the constructed and filled cassette. help.

Hole 268 has a larger opening 270 and a smaller opening 272 that creates a shoulder 274 as best shown in FIG. 7D. The smaller opening 272 has a taper 276. The capture gel 40 covers or overlies the hole 268 for capture of target molecules as described below. As best seen in Figures 7B and 7D, the teeth 218 are tapered such that each of the teeth is narrow and short at this bottom, as compared to the top of the teeth near the handle.

In one embodiment, the capture gel holder 220 has a length of just over 4 inches and has six teeth 218. The centerlines of adjacent holes 268 are 0.709 inches apart and the spacing between the centerlines of the first and last holes 268 is 3.543 inches. The teeth 218 are tapered and have a bottom of 0.520.
The top of the tooth near the handle portion is 0.545 inches long, and 0.138 inches, and 0.138 inches long and 0.08 inches thin at the central region 266.
It has a central area width of inches.

In one embodiment, the holes 268 have a diameter of 0.260 inches. Hole 2
The top of 68 is 0.381 inches above the bottom of the tooth.

In one embodiment, the migration channel 96 has a width of 0.295 inches,
This is larger than the diameter of the hole 268 in the tooth 238.

Slotted side 240 and square side 24 for tooth 238.
4A and 4B with 2, the teeth 218 of the capture gel holder 220, as shown above for the evaporation cover 194, are rounded, as best seen in FIGS. 7B, 7F, and 7G, and 7C. A side edge 280 and a square side edge 282. In addition, some of the teeth 218 are captured by the capture gel holder 2
Additional protrusions 284 may be included on the side edges 282 of the square to ensure that the 20 is placed in the electrophoresis cassette 190 in the proper orientation. 6A, 7A, and 7F show such protrusions. As mentioned above, the teeth 238 are tapered, so that the rounded and square ends 280 and 282, via the slots 238 of the evaporative cover 194, will likely fit incorrectly at a particular distance. obtain. This protrusion 284 limits such a possibility.

Referring back to FIGS. 5 and 6B, the capture gel holder 220 has flat optical detection surfaces 288 at both ends of this portion. This optical detection surface 288 is used with the electrophoretic instrument 292 described in connection with FIG. Although the capture gel holder 220 can be placed in the cassette 190 in only one position, the optical detection surface 288 is located at both ends of the capture gel holder due to the two positions in the electrophoretic instrument 292 that receive the electrophoresis cassette 190. Will be placed.

In addition to the capture gel holder 220, a sample well forming comb 380 as shown in FIG. 8A and a molding comb 386 as shown in FIG. 8B are used with the electrophoresis cassette 190. The sample well-forming comb 380 as shown in FIG. 8A includes a plurality of square holes 236 that pass through the square openings 236 in the evaporation cover 194 to create sample wells in the agarose or electrophoretic matrix 120 as described below. Of teeth 382. Molded comb 386 as shown in FIG. 8B is received in expansion slot 98 during pouring of agarose or electrophoretic matrix 120 as described below.

As mentioned above, the method and apparatus allow for rapid detection of target molecules in biological samples. Capture gel 4 using the described electrophoresis cassette 190
0 and a more detailed description of the electrophoresis cassette 190, a detection method using these features will be described.

METHODS As described herein and illustrated in FIG. 9B, target nucleic acid (DNA
Or a biological sample suspected of containing RNA) is obtained and processed in a manner that releases the nucleic acids and makes them suitable for detection (eg, lysing cells). In one embodiment of the method of the invention, the released nucleic acid is labeled (or tagged) by contacting it with a reporter probe under conditions suitable for the reporter probe to hybridize with the target nucleic acid in the sample. (Eg, detectably labeled) sample nucleic acid is produced. If desired, adapter probes can also be contacted with the sample nucleic acid under suitable conditions,
Here the adapter probe also hybridizes to the sample nucleic acid.
Thus, the sample now contains a mixture of labeled nucleic acids, unlabeled nucleic acids, and other components.

The sample mixture is then subjected to electrophoresis in an electrophoresis cassette as described herein, where the mixture is contacted with a capture probe. The capture probe is specific to the target molecule (eg, target bacterial nucleic acid) contained in the sample. If the sample contains the target molecule (eg, the nucleic acid has a sequence that is complementary to the sequence of the covalently bound capture probe), the target molecule is immobilized on the capture probe in the thin gel of the gel holder. Due to the continuous application of the electrophoretic current, the unbound components of this sample continue to migrate through the thin layer gel and are separated from the bound components. In another embodiment of the method of the invention, gel holders containing immobilized target / capture probe complexes may be transferred to different slots in the electrophoresis cassette and the gel buffered by applying a short voltage. Can be washed with liquid, resulting in unbound (eg, unhybridized)
The components are removed from the gel. After separation, this binding component can be detected by many known methods. In one embodiment, detection of bound target molecules is facilitated by moving the thin layer gel from the electrophoretic device to a detection device (eg, fluorescent or luminescent reader).

FIG. 9A shows the molecular flow in this assay. FIG. 10 outlines the steps for making this sample and reagent. Referring to the blocks of letters in FIGS. 9A and 9B, and block 150 in FIG. 10, a sample suspected of containing the target analyte is prepared. Typically, the molecules detected are cells, bacteria,
Alternatively, it is a target nucleic acid contained in the virus. For example, as shown in FIG. 9C,
Conservative bacterial nucleic acid sequences can be used to detect various bacteria present in biological samples. As described herein, two probes can be used for each target RNA. One is a reporter probe, which is used to label the target RNA. The other is a capture probe, which is linked to a thin gel membrane. Figure 9C shows the SRP RNA sequences from a panel of target organisms (SEQ ID NOs: 1-17) and the complement of this probe sequence. The line surrounded by a thin box indicates the DNA sequence complementary to this probe (the sequence complement is
Mismatched regions are shown so that they can be more easily identified). The white dotted line is
The sequence is underlined corresponding to the capture probe linked to the thin gel membrane. The solid white line is
The sequence is underlined corresponding to the reporter probe. The capture probe is 5'-Ac
rydite (tm) modification is used to synthesize like 2'-O-methyl RNA oligonucleotides (Acrydite (tm) is Mosaic Tech.
commercially available from Nologies, Waltham, MA). The reporter probe is synthesized with a 5'-amino modification, like a 2'-O-methyl RNA oligonucleotide, which is conjugated with an alkaline phosphatase enzyme label.

One of the first steps in sample processing is to lyse the cells so that the nucleic acids are available for hybridization to the capture probe. For example, the sample is placed in a tube with a buffer or diluent and the cellular material (eg,
Platelets, cells, bacteria, viruses) are centrifuged at a speed and for a time sufficient to pellet them. Such techniques are well known to those of ordinary skill in the art. The supernatant is drained from the sample in the tube and the sample resuspended in rinse buffer and then spun down again as before. After centrifugation, drain the supernatant. The lysis buffer is then pipetted into the tube with the sample, as indicated by block 132 in Figure 9A. Lysis buffers include buffered solutions containing sufficient components to lyse cellular material and preserve target nucleic acids in their undegraded state. For example, the lysis buffer can optionally include a detergent or chaotropic agent to facilitate lysis. Such buffers and components are well known to those of skill in the art.

Referring to block 152 in FIG. 10, this sample is, for example, about 1
Heated above 00 ° C, more specifically to about 124 ° C ± 4 ° C for a sufficient time for cells to lyse (eg, about 5 minutes), then at a specific temperature below 50 ° C (eg, 45 ° C) Until about 2-6 minutes. In one embodiment, the sample is heated in the incubator device 302 of FIG. The reporter probe mixture is then introduced into the lysed sample. In one embodiment, the reporter probe comprises an oligonucleotide conjugated to a fluorescent label, phosphorescent chemiluminescent label, or enzyme label. Some examples of enzyme labels include horseradish peroxidase or alkaline phosphatase. Generally, an enzymatic label that produces a fluorescent or chemiluminescent signal is used. This reporter probe is shown in blocks 134 of FIGS. 9A and 9B, and FIG.
Under conditions sufficient to allow the reporter probe to hybridize to the nucleic acid in the sample, as exemplified by block 154 of FIG. , Is contacted with the lysed sample. For example,
One reporter probe known to those of skill in the art is the alkaline phosphatase reporter probe. Accordingly, block 136 of FIGS. 9A and 9B, and FIG.
This sample hybridizes to the reporter probe, yielding a labeled sample, as indicated by 156 of 0.

Referring to block 158 of FIG. 10 and block 138 of FIGS. 9A and 9B, the labeled sample is the electrophoresis cassette 19 shown in FIGS.
Transferred to 0 sample wells. A voltage is applied to the electrophoresis cassette 190 using the capture gel holder 220 located in the set of expansion slots 98 closest to the positive (+) electrode of the migration channel 96 to block 140 of FIG. 9A and block 160 of FIG. As illustrated by, the labeled sample is
From the position introduced into the migration channel 96 away from 00 towards the other electrode 100 through the electrophoretic matrix 120. In some embodiments, the cassette is pre-warmed to a temperature of about 27-35 ° C prior to electrophoresis. In one preferred embodiment, the electrophoresis temperature is about 31-35 ° C. In the preferred embodiment, the voltage is regulated between about 24 and about 40 volts.

The capture probes / ligands can all have the same sequence or can have different sequences. It will be appreciated that each sequence in the lamina gel may be localized to a particular region (eg, a particular region of a particular tooth) or a spatially defined region within a single tooth. Thus, each tooth of the capture gel holder may have a different capture probe / ligand for a different target molecule, or each tooth may contain multiple species of capture probes. In the case where multiple probes are present, each probe may be in a spatially uniquely delineated region of the gel. Alternatively, the capture probes are mixed together and the mixture is dispersed throughout the capture gel.

After a set time, the voltage to the electrophoresis cassette 190 is shut off and the capture gel holder 220 is moved to another enlarged segment 98 (often referred to as a wash slot). This voltage is restarted, and electrophoresis is allowed to
For example, 5 minutes). The current flow is in the same direction during the second or wash period. By moving the capture gel holder 220 to the second enlargement segment 98, the sample migration moves away from the capture gel 40 and thus the capture gel holder 220.
Any unbound sample above will pass through and no new sample will be in contact with capture gel 40. Only the specifically bound target remains on the comb.

Referring to block 142 of FIGS. 9A and 9B, and block 162 of FIG. 10, the capture gel holder 220 (eg, the comb shown in FIG. 5) is removed from the electrophoresis cassette 190 and labeled target nucleic acid. The capture gel 40 including
It hybridizes to the capture probe of the capture gel matrix. The capture gel holder 220 is then placed in a buffered solution and the substrate specific for the reporter probe of the labeled sample has sufficient time for the reporter probe to interact with the substrate and produce a detectable signal. And at temperature, the sample is contacted.
The capture gel holder 220 is then positioned so that a signal (eg, chemiluminescence) is detected, as shown in block 164 of FIG. The presence of a detectable signal is an indication of the presence of the target molecule (eg nucleic acid) in the sample.
This detectable signal is generated by the label of the reporter probe, which is the probe shown in block 134 of Figure 9A or block 154 of Figure 10.

Samples Any biological sample can be analyzed using the methods and devices described herein. The methods of the invention are useful for the analysis of any chemical that is electrophoresed (eg, charged molecules that have detectable mobilities when placed in an electrophoretic field) and that binds to or can be bound by nucleic acids. Applicable. Such substances include, for example, DNA or RNA samples, nucleic acid binding proteins, and aptamer binding partners (aptamers are specific binding partners such as peptides, proteins, drugs, polysaccharides, and small organic molecules). Nucleic acids selected to bind to (eg, theophylline and caffeine) (J
Eison et al., Science, 263: 1425-1429 (1994)).
. For example, the methods described herein use immobilized capture probes to
It can be used for the analysis and purification of target nucleic acids, where specific binding involves base pairing interactions between the sample nucleic acid and the capture probe, as in nucleic acid hybridization. The methods described herein are also useful for purifying sequence-specific nucleic acid binding proteins. This is because synthetic nucleic acids of defined sequence can be immobilized on matrices commonly used for protein electrophoresis.

The test sample can be from any source and can include any molecule capable of forming a binding complex with the capture probe. Specifically included by the present invention is a sample from a biological source containing cells or platelets, a sample obtained using known techniques, body tissue (eg skin, hair, viscera), or body fluid. (
For example, samples derived from blood, plasma, urine, semen, sweat). Other sources of samples suitable for analysis by the methods of the invention are microbiological samples (eg viruses, yeasts and bacteria); plasmids, isolated nucleic acids and agronomic sources (eg recombinant plants). ).

The test sample is treated in a manner known to those of skill in the art such that the target molecule contained in the test sample becomes available for binding or hybridizing. For example, if the target molecule is a nucleic acid present in a cell, a cell lysate is prepared and a crude cell lysate is analyzed, including the target nucleic acid and other cellular components (eg, proteins and lipids). Can be done. Alternatively, the target nucleic acid can be isolated prior to analysis (the target nucleic acid is substantially free of other cellular components). Isolation can be accomplished using known laboratory techniques. The target nucleic acid can also be amplified (eg, by polymerase chain reaction or ligase chain reaction techniques) prior to analysis.

Probes A capture probe is a molecular sequence complementary to a putative analyte that is immobilized on an electrophoretic gel matrix by covalent attachment to a polymer suitable for use as a ligand or electrophoretic medium. Various capture probes can be used in the methods of the invention.
Typically, the capture probe of the present invention comprises a nucleic acid having a nucleotide sequence that is substantially complementary to the target molecule, where the target molecule hybridizes to the capture probe. The complementarity of the nucleic acid capture probe need only be sufficient to specifically bind the target molecule and to demonstrate the presence or absence of the target molecule. Suitable probes for use in the present invention include mixed classes of chimeric probes (eg, peptide nucleic acids) that include RNA, DNA, nucleic acid analogs, and nucleic acids having other organic moieties. The capture probe may be a single-stranded nucleic acid or a double-stranded nucleic acid.

As defined herein, the term “nucleic acid” includes DNA or RNA. Nucleic acid, referred to herein as "isolated," refers to a nucleic acid that has been separated from its source components (eg, in a mixture of nucleic acids such as cells or libraries). (If present) and may be subject to further processing. Isolated nucleic acids include nucleic acids obtained by methods known to those of skill in the art. These isolated nucleic acids include substantially pure nucleic acids, nucleic acids produced by chemical synthesis, nucleic acids produced by a combination of biological and chemical methods, and isolated recombinant nucleic acids. .

“Substantially complementary,” as defined herein, means that the nucleotide sequence of the capture probe need not reflect the exact nucleotide sequence of the target molecule,
It means that the sequence identities must be sufficiently similar to hybridize with the target molecule under the specified conditions. For example, non-complementary bases or additional nucleotides can be interspersed in the sequence, provided that the sequence has sufficiently complementary bases to hybridize together.

The particular conditions for hybridization can be determined empirically by one of ordinary skill in the art. For example, stringency conditions should be selected that significantly reduce non-specific hybridization reactions. The stringency conditions for nucleic acid hybridization are Current Protocols in Mol.
electrical Biology, Ausubel, F.M. M. Et al., Vol. 1
, Addendum, 26, 1991, the teachings of which are incorporated herein by reference in their entirety. Factors such as probe length, base composition, percent mismatch between hybridizing sequences, temperature, and ionic strength affect the stability of nucleic acid hybrids. Stringent conditions, such as moderate or high stringency, can be determined empirically and depend in part on the characteristics of the probe and target molecule.

Typically, the length of the capture probe is at least 5 nucleotides in length, more typically between 5 and 50 nucleotides, and can be as long as thousands of bases.

Probes containing modified nucleotides may also be useful. For example, nucleotides containing deazaguanine and uracil bases can be used in place of guanine and thymine containing nucleotides to reduce the thermostability of hybridized probes (Wetmur, Critical Reviews i.
n Biochemistry and Molecular Biology
, Vol. 26, 227-259, 1991). Similarly, 5-methylcytosine can be substituted for cytosine if a hybrid with increased thermostability is desired (Wetmur, Critical Reviews in Bioc.
Chemistry and Molecular Biology, vol. Two
6, 227-259, 1991). Modification of ribose sugar chain (for example, 2'-O
-Addition of a methyl group can reduce the nuclease sensitivity of immobilized RNA probes (Wagner et al., Nature, 372: 333-335, (1994).
)). Modifications that remove the negative charge from the phosphodiester backbone can increase the thermal stability of hybrids (Moody et al., Nucleic Acids Res.
, 17: 4769-4782 (1989); Iyer et al., J. Am. Biol. Che
m. , 270: 14712-14717 (1995)).

Nucleic acid analogs may also be useful as immobilized probes. One example of a useful nucleic acid analog is peptide nucleic acid (PNA), where standard DNA bases are attached to a modified peptide backbone containing repeating N- (2-aminoethyl) glycine units (Nielsen). Et al., Science, 254: 1497-1500 (1
991)). The peptide backbone is capable of folding bases at precise distances to base pairs with standard DNA and RNA single strands. The PNA-DNA hybrid duplex is much stronger than the equivalent DNA-DNA duplex, preferably due to the fact that there is no negatively charged phosphodiester linkage in this PNA strand. Furthermore, due to its unusual structure, PNA is very resistant to nuclease degradation. For these reasons, PNA nucleic acid analogs are useful in fixed probe assays. It will be apparent to those skilled in the art that similar design strategies can be used to construct other nucleic acid analogs with useful properties for immobilized probe assays.

A “label” as used herein is any means to identify a capture probe bound by an analyte. For example, the label can be a molecule complementary to the capture probe-analyte complex, where the label is a radioisotope, a fluorescent tag,
It is provided with a chemiluminescent tag, or similar chemical tag, such as those used in sandwich hybridization assays. Alternatively,
The label can be an enzyme that can interact with the substrate to produce a detectable signal. The signal probe can be used to provide a label on the capture probe-analyte complex.

If desired, each capture probe in the lamella gel or polymer gel is localized to a specific area. Multiple thin layer gels with different capture probes / ligands
All probes in a particular region can be arranged to form an array so that they have a known sequence. Laminar gels or polymer gels are described further below.

Methods have been developed for covalently attaching capture polymers to polymerizable chemical groups. For example, methods for covalently attaching nucleic acids to polymerizable chemical groups are described in US Pat. No. 5,932,711 (Boles); Rehman et al., Nucleic Ac.
id Res. 27: 649-655 (1999); U.S. Patent Application No. 08/9
71,845 (filed Aug. 8, 1997); U.S. patent application Ser. No. 09 / 285,3.
No. 80 (filed April 2, 1999); U.S. Patent Application No. 09 / 286,091 (
Filed Apr. 2, 1999); US Provisional Patent Application No. 60 / 151,267 (199)
Filed Aug. 27, 1997); and US Provisional Patent Application No. 60 / 177,844 (20
Filed January 25, 2000), the disclosures of which are incorporated herein by reference in their entirety (Kenney, Ray, and Boles, Bio.
See also Techniques 25: 516-521 (1998), the disclosure and teachings of which are incorporated herein in their entirety). The capture probe is also US Provisional Patent Application No. 60 / 151,267 (filed August 27, 1999), US Provisional Patent Application No. 60 / 177,844 (filed January 25, 2000).
, And US patent application Ser. No. 09 / 649,637, entitled "Abrams et al.
Methods of Immobilizing Ligands on S
old Supports and Apparatus and Meth
ods of Use Therefor "(filed August 28, 2000), the teachings of which are covalently attached to the matrix using thiol attachment chemistry, as described in US Pat. obtain. The capture probe is attached to a polymeric material that provides a sieving function to increase contact with the capture probe. The thin gel matrix may also include spacer molecules and molecules that strengthen the matrix.

Nucleic acids, modified nucleic acids and nucleic acid analogs can also be attached to agarose, dextran, cellulose and starch polymers using cyanogen bromide or cyanogen chloride activation. A polymer containing a carboxyl group is a carboximide (ca
It can be attached to a capture probe provided with a primary amine group by rboimide) coupling. Polymers with primary amine groups incorporated into the polymer can be attached to amine-containing capture probes with glutaraldehyde and cyanogen chloride. Many polymers can be modified with thiol reactive groups that can be attached to thiol-containing synthetic capture probes. A representative protocol and other examples are C
ass and Ligler (ed.) "Immobilized Biomolec"
ules in Analysis: A Practical Approac
h ”1998, Oxford University Press, Oxford
d, UK, and Hermanson, Mallia, and Smith "I.
Mmobilized Affinity Ligand Technique
s "1992, Academic Press, San Diego, CA, the disclosures of which are incorporated by reference.

Adapter probes / molecules can also be used in the methods of the invention. Such probes are described in US patent application Ser. No. 09 / 285,380 and PCT / US00 /
08529 (the teachings of which are incorporated herein by reference).

Gel Matrix One element of the present invention is a capture gel 40 (eg, a polymer gel or lamella gel). This capture gel matrix is used in combination with a probe or capture probe to capture the target molecule (ie the desired component).

Capture gel refers to the framework of the gel to which complementary sequences bind. Examples of materials suitable for use in forming the gel matrix include both gel-forming and non-gel-forming polymers. Any gel matrix suitable for electrophoresis can be used to prepare the gels of the invention. Suitable matrices include acrylamide and agarose. It will be appreciated that other materials may also be used for the capture gel. Examples include chemically modified acrylamide, starch, dextran, and cellulose-based polymers. Further examples include modified acrylamide and acrylate esters (eg, Polymers, Inc. Polymer & Mono).
Mer Catalog, 1996-1997, Warrington, PA), Starch (Smithies, Biochem. J., 71: 585 (Smithies, Biochem. J., 71: 585.
1959); product number S5651, Sigma Chemical Co. , S
t. Louis, MO), dextran (eg, Polysciences,
Inc. Polymer & Monomer Catalog, 1996-1997
Warrington, PA), and cellulose-based polymers (eg, Quesada, Current Opinion in Bi;
technology, 8: 82-93 (1997)). Any of these polymers listed above can be chemically modified to allow specific attachment of capture probes for use in the invention. Other suitable methods can be found in the literature (for review, see Wong “Chemist”).
ry of Protein Conjugation and Cross-
linking "CRC Press, Boca Raton, FL. , 199
See 3). Incorporating reinforcement of the thin gel matrix facilitates handling of the capture gel.

Hybrid matrices of polymers are also useful in forming the lamellar gels of the present invention. A hybrid matrix comprises a mixture of two or more matrix-forming materials (
For example, acrylamide-agarose mixed gel). These gels typically contain 2-5% acrylamide and 0.5-1% agarose. In these gels, the concentration of acrylamide determines the functional pore size. Agarose provides mechanical strength without significantly changing the gel pore size of acrylamide.

Referring to FIG. 11A, the capture gel 40 has a non-conductive mesh 42 encased in a polymer gel 44. A plurality of capture probe molecules are covalently attached to the polymer gel 44 through the three-dimensional capture gel, which fills the spaces 48 of the fibers 50 of the mesh 42, as best seen in FIG. 11B. The non-conductive mesh 42 is believed to be substantially trapped in the bubble-free trap (thin layer) gel 40. In the cross-sectional view of FIG. 11B, the top surface 52 and the bottom surface 54 of the capture gel (reinforced lamella gel) 40 are shown to be substantially flat. The top surface 52 and the bottom surface 54 are shown as being substantially coplanar with the top surface 52 and the bottom surface 54 of the non-conductive mesh 42, respectively, although either the top surface 52 or the bottom surface 54 of the capture gel 40. Either or both may be spaced from each surface of the mesh 42.

In one embodiment, polymer gel 44 is an acrylamide gel.
The type of capture probe molecule depends on what is desired to be captured. For example,
The capture probe molecule can be an oligonucleotide bearing a reactive ethylene group.

The capture gel 40 with the non-conductive mesh 42 can be formed by various methods. One method of forming the capture gel 40 is by capillarity. A non-conductive mesh of porous material is placed between two glass plates so that the material extends between the two plates while being supported by the one plate.
The gel forming solution is applied to the edge of the material extending from under the top glass plate. The gel-forming polymer solution is drawn to this material by capillarity and between two glass plates. Plate spacers can be used between the glass plates, allowing an increase in the thickness of that layer when gelled. Preferably, a release agent is applied to the mold (which is glass or some other material) to facilitate the release of the shaped and reinforced lamina gel.

An alternative method involves a porous material placed in a mold and gel forming polymer solution (a ligand covalently bound to the polymer that provides the gel with sieving properties).
Is added to the mold to allow solidification. Air bubbles in the gel matrix should be avoided, for example, by forming a thin gel under pressure or by vibrating the mold.

Referring now to FIGS. 12A and 12B, the reinforced capture (thin layer) gel 5 with the polymer gel 44 encased within the fibrous glass fibers (below structure 60).
Eight alternative embodiments are shown. It can be seen that the glass fiber reinforcement 60 extends above and below the surface of the polymer gel 44. The capture probe molecules are covalently attached to the polymer 44 through the three-dimensional capture gel 58.

Preferably, the capture gel 40 of the present invention has sufficient mechanical strength to allow it to be removed from the electrophoretic device for washing and detection procedures (eg, described below). . However, the capture gel can be reinforced by either mechanical or molecular means. Examples of such internal structural reinforcement of thin gel matrices include various non-conductive materials having pores. Such materials include, for example, mesh, screen or honeycomb materials; non-woven fibrous materials (eg glass fibers and mats); and woven or knitted materials (eg woven materials). Materials having holes include mesh materials, woven materials and non-woven materials. Examples of the mesh material include polyester silk screening materials, for example, Separ America, Inc. , Briancliff
Manor, NJ. Corp. , Polyamide resin mesh, nylon mesh, and polyethylene or polypropylene honeycomb material. As a woven material, a non-fast woven material (for example, John
son & Johnson Ltd. J-Cloth ^™, etc., available from Nonwoven materials include cellulose acetate butyrate, nitrocellulose, cellulose propionate (see, for example, US Pat. No. 4,006,069 (Hirat).
suka et al.), the disclosures of which are incorporated herein by reference), including membranous materials including cellulose esters. If the reinforcement is inside a thin gel matrix and there is a gel layer on both sides of the reinforcing mesh or cloth so that the reinforcing material is wrapped by the gel and entangled with the gel, the thickness of the gel is Preferably, it is no larger than what can be supported by the mesh or cloth. For embodiments in which the gel is retained primarily on the surface of the reinforcement material, the surface of the reinforcement material may be rough, irregular, porous, or else the gel may penetrate this material. It is preferably either possible. Some examples of three-dimensionally stable reinforcing materials are porous materials such as GB 142.
No. 1531 (UKEAA), the disclosure of which is incorporated herein by reference in its entirety, are porous particles made by a method such as that disclosed in US Pat. Other examples of reinforcing materials include thinly-divided sponges. Another contemplated enhancement is US Pat. No. 5,672,416 (Radola) (this disclosure is
Comprising a porous material that is coated to promote adhesion of the gel matrix to the porous material (eg, coated polyester, as described in its entirety herein). ).

The capture gel 40 may also include spacer molecules and matrix enhancing molecules. As used herein, a "spacer molecule" is a structure that is capable of polymerizing with or attached to at least one gel matrix-forming polymer molecule and is the portion of the gel matrix-forming polymer backbone that is attached to the spacer. Function to space the. Optionally, the spacer molecule can be attached to any two gel matrix-forming polymer compounds, and thereby the density of capture probes attached to the gel matrix-forming polymer component in any particular region of the lamellar gel matrix. It can be effected by spacing one polymer from another polymer in the matrix in a spatial and / or spatial direction. In addition, the spacer molecule may provide reactive sites for attachment to moieties or molecules other than the matrix-forming polymer. For example, the spacer molecule may provide a binding site for polyethylene glycol or a surfactant.

As used herein, a “matrix-reinforced polymer” means to crosslink, stiffen, or even crosslink without adversely affecting the desired sieving and / or analyte binding properties of the resulting thin gel matrix. If not, it is a useful polymer for modifying the gel matrix to make it more durable during operation. The matrix-reinforced polymer hardens the thin gel matrix and increases its mechanical strength, thus allowing manipulation of this matrix without further external reinforcement. An exemplary polymer includes agarose. In addition to reinforcing the lamellar gel matrix, the polymeric component may also provide additional sites for coupling gels that modify the composition. For example, chemical groups such as methyl or hydroxyl groups may be added to modify the hydrophobic / hydrophilic properties of the gel. Sulfate or quaternary amine groups can be added to introduce ionic groups into the thin layer gel.

Methods of Preparing Cassettes With respect to the electrophoresis cassette 190 briefly described, the assembly process of the electrophoresis cassette will be described in more detail. Stainless steel or platinum strips 206 are placed in the electrode channels 92 and 94 found in FIG. The strip 206 has curvilinear ends, receives the post 204 as shown in FIG. 3 as described below, and the slot 200 in the rib 198.
Ensures that this strip is installed correctly.

With the electrode strip 206 in place, an evaporation cover 194 as found in FIGS. 4A and 4B is disposed on the base 192. As shown above,
Cover 194 and base 192 have tabs 248 to ensure accurate installation.
And notch 250. In one embodiment, evaporation cover 194 and base 192 are welded together by an ultrasonic process.

The four posts 204 associated with the two electrodes 202 are each provided with an evaporation cover 194.
It is inserted into one of the holes 246 therein. As can be seen in FIG. 1, the base 19
The two holes 210 receive a portion of the syringe rod portion of the post 204, which has a rod portion that passes through the curved end of the strip 206. An annular disk portion 212 of post 204 overlies the top of evaporation cover 194.

Evaporation cover 194 and base 192 of electrophoresis cassette 190 assembly
With respect to, the pair of molding comb teeth are provided with an evaporation cover 19 as shown in FIG.
4 through slots 238 and enter expansion slot 98. One of the same forming combs, 386, is found in Figure 8B. The expansion slots 98 of both sets receive the teeth of the forming comb. Instead of using the pair of forming combs 386 of FIG. 8B, a single forming comb 390 with two parallel sets of teeth as found in FIG. 8C can be used.

With the teeth of the shaped comb 386 or 390 in place in the enlarged slot 98 of the transfer channel 96, the electrophoretic matrix 120 is dispensed through the sample opening into the base 192 and moved to a predetermined height. Channel 9
Fill a portion of 6. In one embodiment, electrophoretic matrix 120 may be formed from a gel-forming polymer such as agarose or starch. In one embodiment, agarose is formed with a forming solution of 1% agarose in 0.1 × TrisBP. The following is mixed: 100 ml 0.1
X TrisBP buffer and 1 g agarose powder (Sigma A9539 Gen
(Enal Use Molecular Biology Grade). This procedure is repeated for all migration channels 96 and the sample well comb 380 is inserted into the cassette. The comb of the sample well is shown in Figure 8A.

Note that the shaped agarose solution ensures a sufficiently empty space to allow the insertion of the sample well comb, and the sample opening 23 in the evaporation cover is
6 to channels 96.

For illustration purposes, an agarose or electrophoretic matrix 120 filling one migration channel is shown in FIG. Sample well 174 is shown in agarose 120. As indicated above, the evaporation cover 194 is welded to the holes 192 prior to dispensing the agarose 120 into the channels 96.

For the embodiment shown in FIGS. 1-3, about 1.4 m. 1 agarose solution
Used per channel. After fitting all six channels 96, FIG.
The sample well comb shown at is inserted into the sample opening 236 and the agarose is cooled. After cooling, the mold and sample well combs are removed and the sample wells 174 in electrode channels 92 and 94 and channel 96 are filled with electrophoresis running buffer through cover openings 238 and 236, respectively. In addition, wash buffer is placed in wash well 222 and conditioning buffer is placed in conditioning well 224.

With respect to the agarose in the electrophoresis cassette 190 with the sample wells formed and the appropriate buffer in place, the electrophoresis cassette 190 was foil wrapped and sealed for transport in one embodiment. Can be one. The capture gel holder 220 is separately sealed and, in one embodiment, contains the desired capture probe.

The cassettes herein can be disposable (ie, used once and discarded) or reused after a suitable cleaning process. For example, in an embodiment utilizing alkaline phosphatase (AP) as a label,
Post-treatments to remove traces of AP can be used. A portion of the electrophoresis cassette 190 is submerged in a solution of 1% hydrochloric acid and 1 millimolar (mM) EDTA (ethylenediaminetetraacetic acid) for at least 30 minutes to remove residual reagents. Part or components are then thoroughly washed with water and air dried on a clean surface.

If detection of the target molecule is desired, the electrophoresis cassette 190 and the capture gel holder 220 will be opened if the sealing step had occurred. The sample is also shown in FIG.
Prepared as described with reference to A, 9B, and 10. The hybridization mixture is loaded into the sample well 174 in the electrophoresis cassette 190 through the square opening 236 in the evaporation cover 194.

The capture gel holder 220 containing the capture gel 40 with the desired capture probe is
Inserted in the electrophoresis cassette so that the teeth 218 enter the first set of expansion slots 98 in the migration channel 90. The first set of this slot 98 is (
The set closest to the (+) electrode.

The electrophoresis cassette 190 is placed in the electrophoresis machine 292. Referring to FIG. 13, an electrophoretic machine 292 for receiving the electrophoretic cassette 190 is shown. In FIG. 13, one side of the electrophoretic machine 292 has been moved for illustration purposes. The electrophoresis machine 292 has a tray 294 that slides out of the machine to receive the electrophoresis cassette 190. This machine 292 electrically connects to the center of the electrophoretic machine 292 at bar 296 so that when the tray 294 slides into the machine 292, the center bar 296 will cause the syringe disc of the post 204 of the electrode 202 to be flush. Mesh with 212. Only one side of each electrode 202 is touched. The purpose of having a pair of posts 204 for each electrode 202 is that the electrophoretic cassette 190 can be placed in any spot on tray 294 in electrophoretic machine 292.

The electrophoretic machine 292 has temporal features and temperature sensors to ensure that the electrophoretic cassette 190 does not exceed a certain temperature. Further, with respect to the capture gel holder 220, an optical detection surface 288 is used in combination with the electrophoretic machine 292 so that the machine 292 does not operate without the capture gel holder 220.

Referring to FIG. 14, an electrophoretic machine 292 is shown with an overlying incubator unit 302. The incubator unit 302 has a pair of heating blocks 304 for receiving the electrophoresis cassette 190. Block 304 is used to preheat cassette 190 to the appropriate electrophoresis temperature prior to electrophoresis.

The electrode 100 of the electrophoresis cassette 190 of FIG. 2 is a DC in the electrophoresis machine 292.
It is connected to a power source and a constant voltage is applied for a constant time. The temperature increases during electrophoresis, but should not be allowed to increase above that which impairs hybridization of the probe with the target nucleic acid. In the preferred embodiment, the temperature of the cassette is controlled within the electrophoresis machine 292.

Referring to block 158 of FIG. 10, the capture gel 4 in the capture gel holder 66.
After running the sample on the electrophoretic matrix 120 toward 0 for a certain period of time, the power is turned off. This capture gel holder 66 is used for the expansion segment 9 in the migration channel 96.
From 8 to another enlarged segment 98 in the same electrophoresis cassette 86 proximal to the (-) electrode. Prior to transfer to another enlarged segment 98, the teeth of the capture gel holder 220 are placed in a set of wash wells 222 containing wash buffer. 1
In one embodiment, the buffer is the wash buffer of Example 1, and
The capture gel 40 on the tooth 218 is in the buffer for 5 seconds.

The electrode 100 is connected to a DC power supply, and a constant voltage is applied for a constant period. Again, be careful that the temperature of the cassette does not exceed the temperature that would compromise probe hybridization.

As discussed above, when discussing the method related to FIG. 10, the current is in the same direction during the second period or wash period. Capture gel holder 2 in second enlarged segment 98
By moving 20, the propagation of excess sample that may be trapped in the channel 96 moves away from the capture gel 40 of the capture gel holder 220, thus allowing any sample on the capture gel holder 220 that is not captured by the capture probe to pass through. And, there are no new samples in contact with the capture gel 40.

After the above steps, detection of the capture nucleic acid target alkaline phosphatase-conjugated probe complex on the thin gel film, the capture gel 40 of the capture gel holder 220, is achieved. The capture gel holder 220 is removed from the wash or second set of slots 98 of the electrophoresis cassette. The tooth 218 with the capture gel 40 is
24 in a second set of conditioning buffer. The capture gel 40 is immersed in the solution and then incubated at room temperature for 5-10 minutes on the incubator unit of FIG.

Referring to FIGS. 15A and 15B, a detection tray 178 for receiving the capture gel holder 220 is located on a detection device 172 such as the luminometer of FIG.
The detection tray 178 has a recess 31 for receiving a pair of capture gel holders 220.
Have two. In one embodiment, the recess 312 is shaped to fit the capture gel holder 220, as described above. The detection tray 178, in one embodiment, is approximately the size of a 96-well tray plate in length and width. In this embodiment, the detection tray 178 is not as tall as a conventional 96-well tray plate. Therefore, it has a length of approximately 5.03 inches and a width of 3.365 inches.

The capture gel holder 220 is then placed on the detection tray 178, as shown in FIGS. 15A & 15B. The capture gel holder 66 and the detection tray 178 are made of E. coli, as shown in FIG. G. & G Walla of Turku, Fin
It is placed in a microplate luminometer 172 such as the Walla Victor 2 sold by Land. Relative light units (RAU) are read, and RAU, and signal to noise ratio = (test sample membrane-empty well) /
Both (negative control sample membrane-empty well) are reported.

Method of Purification In another embodiment of the invention, target nucleic acids are purified by contacting with a capture probe covalently attached to a thin gel matrix. For example, after the target nucleic acid is bound to the capture probe and separated from the rest of the sample, the bound target nucleic acid can be released from the probe by changing conditions, such as increased temperature, and The released target nucleic acid can be collected in a clean container for further processing. Such purification can be performed using a capture probe covalently attached to a thin gel membrane, where the gel membrane can be any suitable holder or support device (eg, those described herein). Combs as described above) or microliter plates (such as 96 well microliter well plates well known to those skilled in the art).

For example, referring to FIG. 17, the purification step is performed by the method shown in FIG.
The detection process blocks 162 and 164 of FIG. 10 are similar to the method discussed above. The capture gel holder 220 is removed from the electrophoresis cassette 190 after the desired target nucleic acid molecule has been captured and immobilized on the capture probe in the gel. The capture gel holder 220 is subjected to conditions sufficient to break the bond between the capture probe and the target molecule, thereby releasing the target molecule from the probe. In the embodiment shown in Example 17, the thin film is removed from the holder and placed in a tube with elution buffer. The tube is heated to elute the hybridized target from the capture probe. The released target molecule can be recovered using techniques well known to those of skill in the art and then, for example, PC
Used in further procedures such as R amplification.

Alternative Cassettes and Holders An alternative capture gel holder 66 is seen in Figures 18A and 18B.
The capture gel holder is also sometimes referred to as a comb or sample comb, 66 having a handle 68 and a plurality of teeth 70.

Each of the teeth 70 has a lamina central region 74 located in the hole 76. The holes 76 extend through the teeth 70. The lamina central region 74 facilitates bubble removal as described above with respect to the first embodiment. The non-conductive mesh 42 of the capture gel 40 is
Lying on the hole 76.

The capture gel 40 can be applied to the capture gel holder 66 by a number of methods including, but not limited to, ultrasonic welding, heat staking, and gluing. The polymer gel 44 component of the capture gel 40 may be cast onto the non-conductive mesh 43 before or after attaching the mesh to the holder 66. In one embodiment, the capture gel 40 placed on the non-conductive mesh 42 in front of the mesh is adhered to the holder 66.

An alternative electrophoresis cassette 86 for use with the capture gel holder 66 is found in Figures 19A-19C.

Referring to FIG. 19A, the electrophoresis cassette 86 has a base 88 and an evaporation cover 90. The evaporation cover 90 is transparent in one embodiment, but is not so limited.

The base 88 of the electrophoresis cassette 86 has a pair of electrode channels 92 and 94, as shown in FIG. 19B. The base 88 has a plurality of migration channels 96 extending between the electrode channels 92 and 94. Within each migration channel 96 is a pair of expansion slots 98 adapted to receive the teeth 70 of the capture gel holder 66.

Each electrode channel 92 and 94 has an electrode 100 that extends the length of the respective electrode channel 92 and 94. In one embodiment, the electrodes 100 are each a pair of stainless steel pins 108, and the stainless steel strips 110 extend between the pins 108.

The evaporation cover 90 has a strip 102 lying on the interface between the electrode channels 92 and 94 and the migration channel 96. The strips 102 limit the flow of bubbles produced by buffer hydrolysis at the electrodes, thereby facilitating their removal through the vents 104 in the cover. The evaporation cover 90 has a plurality of vent holes 104 that overlie the electrodes 100. In addition, the evaporation cover 90 has holes 106 for receiving the teeth 70 of the capture gel holder 66 which overlie the enlarged slots 98 of the migration channel 96 of the base 88.

In addition, the evaporation cover 90 has a plurality of generally square openings 114. The square openings 114 are sample well forming combs 118, as seen in FIG.
A plurality of teeth 116 of the same are allowed to pass through the migration channel 96, as described below.

Second Alternative Electrophoresis Cassette Referring to FIGS. 21A-21C, electrophoretically sampled into a reinforced thin layer gel (or thin layer gel) with capture probes covalently attached to a thin layer gel matrix. A second alternative electrophoresis cassette 150 for use in the method of introducing a. The cassette 450 includes a base 452, a first electrode 454, and a second electrode 45.
6, including at least one holder 458 for holding the reinforced thin layer gel 406 and an electrophoretic matrix. The electrophoretic matrix is filled with buffer and occupies substantially the cassette base as a layer having a predetermined height.
The cassette is provided with at least one well 460 in an electrophoretic matrix for receiving a liquid sample. The electrophoretic matrix can be formed, for example, from a gel forming polymer such as agarose or starch. Alternatively, the electrophoretic matrix may be prepared according to Harrington et al., US Pat.
37,202, the disclosure of which is incorporated herein by reference in its entirety.
It may be formed from a porous solid such as a polymer sponge, as described in.

The reinforced lamina gel is maintained in the cassette in the proper orientation by the holder 458. In addition, this holder allows easy removal and transport of the enhanced thin layer gel to the detection station for readout of the enhanced thin layer gel, as well as detection of target molecules present in the sample. As shown in FIG. 21C, the holder, capture gel holder, has at least one tooth 459 extending downwardly from the handle. The tooth has an opening for receiving the reinforced lamina gel. The holder is oriented so that when the holder is placed in the electrophoretic system, the teeth of the holder extend downward in the electrophoretic matrix. The holder positions the lamella gel so that when a potential gradient is applied across electrodes 454 and 456, the target molecules in the sample in the wells migrate through the reinforced lamella gel. In at least one embodiment of the electrophoretic system, holders 458 fit into the left and right slots of base 452 to position the holder within the cassette.

The electrodes include wires, carbon rods, metal pieces, metal plates, conductive plastics, etc.,
It can be formed from a number of conductive materials. These conductive electrode materials may be formed as wires, flakes, pins, rods, coatings deposited on the ends of base 452, or conductive adhesive strips.

A preferred method for preparing the cassette of FIG. 21A is described below. First, the electrode and base are assembled. Then, the bonded reinforced thin layer gel 40
A holder 458 with 6 is placed in the base. The teeth of the holder 458 are
Immersed in matrix 550 to a depth indicated by dotted line 601 in FIG. 21C. The comb with the hard teeth is then placed adjacent to and in parallel with the holder 158. Comb 1 with comb handle 182 and teeth 184
An illustrative example of 80 is provided in Figure 8B. A holder 188 having a handle 182, teeth 184, and openings 190 in the teeth for receiving the reinforced lamina gel.
An illustrative example of is provided in FIGS. 18A and 18B. The comb teeth for well formation accept the reinforced lamina gel so that the electric field drives sample molecules straight from the sample well through the reinforced lamina gel past the capture probe in the finished cassette. It is preferably aligned with a tooth having an opening for. Once the comb and holder are placed in the base 452, the base is filled with molten agarose in electrophoresis buffer. The filled cassette is cooled and the agarose solidifies. After cooling, the sample well forming comb is removed. The complete cassette is illustrated in Figure 21A.

A particular embodiment of the invention is a capture comb as described herein, although any holder or manifold capable of supporting a gel matrix may be used in the methods described herein. It is also understood to be suitable for. A key feature of this holder is its ability to stably support the matrix (eg, weld the mesh-coated gel to the holder) in a manner suitable for electrophoresis. For example, numerous devices and manifolds commonly used for filtration and vacuum filtration can be adapted for use as capture gel holders. For example,
Multiscreen ^™ product system (Millipore Corp. Bedfo
rd, MA) see the 96-well manifold of the type used.

The present invention is particularly shown and described with reference to its preferred embodiments,
It will be appreciated by those skilled in the art that various changes in form and detail can be made herein without departing from the scope of the invention, which is included in the appended claims.

Examples Example 1-Assay Procedures Sample Processing 1 mL of sample material (eg, platelet concentrate) was pumped out and 0.5
Dispense into sample vials containing mL sample diluent. The tube was capped and inverted 3-5 times for mixing. Centrifuge the tubes at 10,000 ± 1,000 xg for 1 ± 0.25 minutes in a balanced microcentrifuge. Control tubes are not centrifuged. Carefully remove the tube from the rotor and remove the cap. Pour into a BioHazard waste container to decant the supernatant and briefly keep the rim of the tube open for clean absorbent material. This tube should not be tapped or shaken.

Add 1 mL rinse buffer to each tube, resuspend by gently tapping the tube several times and vortex for 10 seconds or until the pellet is evenly suspended. Recap and place in rack. Incubate the tubes for 10-14 minutes at room temperature. Centrifuge at 10,000 ± 1,000 × g for 1 ± 0.25 minutes in a balanced microcentrifuge. Again, the control tube is not centrifuged. Carefully remove the tube from the rotor and remove the cap. Pour into a BioHazard waste container to decant the supernatant and briefly keep the rim of the tube open for clean absorbent material. This tube should not be tapped or shaken.

Add 50 ± 5 μl lysis buffer to each sample tube. The tube is tightly capped, resuspended by tapping the tube several times, and vortexed for approximately 10 seconds or until the pellet is uniformly resuspended. Negative control (NC) and positive control (PC) tubes are obtained for each electrophoresis. All tubes were placed in a Lysis Unit (heater block) at 45 ° C. Let stand at 45 ° C. for about 5 minutes. Place control and sample tubes in heater 302 and heat to 124 + 4 ° C. Cool the tube to below 50 ° C. The tube now contains a sample lysate suitable for nucleic acid probe assays.

(Hybridization) Before opening the tube, in order to bring most of the liquid from the bottom of the tube,
Shake four times. Add 25 ± 3 μl probe buffer to samples and controls. Cap the tube and mix by shaking 3-4 times to bring most of the liquid from the bottom of the tube. 10 ± 1 minute, 45 ±
Place the tube in a 2 ° C heater. Before opening the tube, shake it 3-4 times to bring most of the liquid from the bottom of the tube. The tube currently contains the sample hybridization mixture ready for electrophoresis. Insert comb into cassette prior to sample loading.

Electrophoresis Load 25 ± 3 μl from each tube into the sample wells of cassette 190 preheated on a block of 31 ± 4 ° C. as described with respect to FIGS.
A new pipette tip should be used for each sample. Voltage 4
Bring to 0 volts, electrophorese for 4 minutes, then reduce voltage to 24 volts for an additional 16 minutes. The voltage was turned off and the membrane comb was transferred to the wash buffer wells and kept in room temperature or preheated cassette for approximately 5 minutes. Move the comb to the electrophoretic wash slot and bring the voltage to 40 volts for 5 ± 0.5 minutes. The voltage was turned off, the membrane comb was transferred to the conditioning buffer and kept at room temperature or preheated cassette for 5-10 minutes.

Detection: The comb is removed from the cassette and blotted on the side of the comb for 10 seconds with clean absorbent material maintained in the blotting equipment. Place the comb in the leader tray and place the leader tray in the leader. 25 ± 3μ
l of room temperature substrate is added to each well and read. Sample dilution buffer: NaH ₂ PO ₄ monohydrate 15 mM Na ₂ HPO ₄ heptahydrate 15 mM EDTA 15 mM sodium dodecyl sulfate (SDS) 0.30% ProClin 5000 0.02% pH 7.0 +/− 0.1 Sample rinse buffer: NaH ₂ PO ₄ monohydrate 2.0 mM Na ₂ HPO ₄ heptahydrate 4.0 mM EDTA 1.5 mM sodium dodecyl sulfate (SDS) 0.30% sodium chloride 34 mM potassium chloride 1 mM ProClin 5000 0 0.02% Phenol Red 0.001% pH 7.0 +/- 0.1 Lysis buffer: NaH ₂ PO ₄ monohydrate 75 mM Na ₂ HPO ₄ heptahydrate 75 mM EDTA 4.5 mM sodium dodecyl sulfate (SDS) 7.50% Dextran (188kD) 10% Luforodamine B 0.001% ProClin 5000 0.02% pH 7.0 +/- 0.1 probe buffer: Tris base 25 mM NaCl 525 mM Ficoll, 70 kDa 5% xylene cyanol 0.05% casein, Hamarsten grade 0.5 mg / mL sodium azide 0.04% MgCl ₂ 0.5mM ZnCl ₂ 0.05mM reporter probe alkaline phosphatase conjugated 2'-0-methyl oligonucleotide (see FIG. 9C for a description of the capture probe and reporter probe) 25 nM pH 8.0 +/- 0.1 Wash Buffer: Tris Base 90 mM Boric Acid 72 mM Phosphoric Acid 5.5 mM SDS 1% Triton X-100 1% ProClin 5000 0.02% Bromphenol Blue 0.0. 005% pH 8.3 +/- 0.1 Conditioning buffer: DEA 100 mM Magnesium chloride 0.1 mM Zinc chloride 0.01 mM ProClin 5000 0.02% pH 10.0 +/- 0.1 Alkaline phosphatase buffer: Tris base 25 mM Chloride Sodium 50 mM Magnesium chloride 1 mM Zinc chloride 0.1 mM Sodium azide 0.04% pH 8.0 +/− 0.1 (Example 2-assay using thin gel membrane) In this example, the cassette of FIG. Similar cassettes were used with the following exceptions: 1) carbon rod electrodes were used instead of metal rods; 2) individual mesh reinforced lamina gel membranes were used instead of multiple tooth capture gel holders. did. Individual thin gel membranes were manipulated with forceps.

Part I. Preparation of Thin Layer Gel Membrane for Capture of Target Nucleic Acids A. Preparation of Mesh A polyester mesh having a thickness of 100 microns is separated by Separ Amer.
ica, Inc. Briaciff Manor, NJ. Available from.
Cut this mesh into rectangles (0.48 inches x 0.52 inches), ethanol (1 wash for 5 minutes), deionized water (3 5 minutes washes), and an aqueous nonionic interface. Wash by gentle shaking in active agent (0.1% Tween 20, 1 wash for 5 minutes). The mesh was air dried on clean filter paper.

B. Preparation of Gel Matrix Solution Mix the following in a 1 ml tube: 337 microliter deionized water; 2
9: 1 ratio of monomer to bisacrylamide (BioRad Laborat)
ories, Richmond, CA) 62 microliters 40% acrylamide aqueous solution; 50 microliters Tris BP buffer (Tris BP buffer is 900 mM Tris borate buffer (TrisBp) pH 8.3 and 900 mM)
Tris phosphate buffer pH 8.3 was mixed at 4: 1); 5'acrylamide modification (5'-acrylamide-AGGCCCGGGAACGTATTCAC-3 '(
SEQ ID NO: 18)) in 50 microliters of 500 micromolar oligonucleotide capture probe (Acrydite ^™ modification,
Mosaic Technologies, Boston, MA); 1 microliter 20% TWEEN 20; and 3 microliters 10% TEMED (
(5 microliters in 45 microliters H ₂ O) C. Polymerization to form a thin gel film A rectangular mesh is sandwiched between clean silanized glass plates. On one end of the sandwich, slightly shift the plate at one end so that the mesh lying against the bottom plate is exposed. To the capture mix prepared in Part B (above), add 4 microliters of 10% ammonium persulfate (
50 mg of water in 500 microliters) is added, mixed and the mixture spotted on the exposed mesh, allowing it to be transferred to the mesh between glass plates. If the mesh is saturated, slide the top plate so that the mesh is completely sandwiched between the two plates. Polymerization was allowed for 1 hour at room temperature. The glass plate was separated using a razor blade, and excess polyacrylamide gel was excised from the rectangular thin gel film. Membranes were washed by immersing them in a large volume of electrophoresis buffer (ie 0.1x Tris BP buffer).

Part II. Cassette Construction The cassette base used in this example was similar to that shown in Figures 19A-19C. Other cassette components include: one sample well homing comb for homing sample wells in channel 301; and two carbon electrode rods wrapped with platinum wire (platinum wire to power source). Present for the connection of carbon rods). These cassette components were treated with 1% hydrochloric acid and 1% to remove residual alkaline phosphatase activity.
Immerse in a solution of millimolar (mM) EDTA (ethylenediaminetetraacetic acid) for at least 30 minutes. These components are then thoroughly washed with water and air dried on a clean surface.

Another set of cassette components is washed and dried including: Figure 19
Cassette base shown in Figure 2; two carbon electrode rods wrapped with platinum wire (the platinum wire is present for the connection of the carbon rod to the power source); and in the cassette slot 98 during capture and cleaning. One sample well homing comb (shown in Figure 20) for homing the wells to hold the membrane in place. This second set is used for the electrophoretic wash of this gel film after target capture.

A solution of 1% agarose in 0.1 × TrisBP was dissolved in 2 × as follows.
Prepare in a 50 ml glass Erlenmeyer flask. 100 ml of 0.1
X TrisBP buffer and 1 g agarose powder (Sigma A9539 G
General use Molecular Biology Grade) is mixed. The head of the flask is loosely stoppered with clean laboratory tissue (Kimwipe) and heated to boiling in a commercial microwave oven. Cover the flask with an aluminum wheel and equilibrate in a water bath at 60 ° C.

(Construction of Sample Electrophoresis Cassette) A carbon electrode was connected to each electrode channel of the sample cassette (92 and 94 in FIG. 21).
Install inside. Pour the dissolved agarose gel solution into the cassette, taking care to allow enough space for the base to not overflow when the comb is inserted. Remove any trapped air bubbles with a pipette tip or a pass rule pipette. Position the carbon electrodes relative to the outer walls within the electrode channels 92 and 94, making sure they are laterally symmetrical with respect to the sample electrophoresis channel.

Insert the sample well homing comb 118 into the slot 124 of the cassette base, making sure the tooth is in the center of the channel and that the comb is not too close to the membrane slot.

One gel film is inserted into each slot 98, making sure that the film is placed against the side of the sample well and that it is centered with respect to the channel. Let the dissolved agarose gel stand for 30 minutes.

(Construction of Wash Cassette (When Separate Wash Cassette is Used)) As described above, two carbon electrodes are placed on the base and the base is filled with the dissolved agarose gel solution. A membrane slot homing comb as shown in FIG. 20 is inserted into slot 98. Check for any bubbles and reposition electrodes as above. Let the dissolved agarose gel stand for 30 minutes.

Part III. Cassette Manipulation Sample Electrophoresis for Capture Remove the sample comb from the sample electrophoresis cassette and fill the wells with electrophoresis buffer (0.1 × TrisBP). Sample wells filled with this buffer are loaded with 20 microliters (μl) of sample. DC from electrode
Connect the platinum lead to the power supply and apply a constant pressure of 50 V for 10 minutes. The temperature increases during the run, but should not exceed 35 degrees Celsius (C). If desired, the cassette can be placed on a surface cooled by a recirculating water bath to prevent overheating.

Electrophoretic Washing of Gel Films The well-homing comb is pulled out of the wash cassette and the slots filled with electrophoresis buffer. Using forceps, pull the membrane out of the sample electrophoresis cassette slot 302 and transfer each membrane to the buffer filled slot in the wash cassette. Removal of the membrane from the sample electrophoresis cassette is facilitated by rocking the membrane back and forth before pulling it up.

The electrodes are connected to a DC power source and a constant pressure of 50V is applied for 5 minutes, taking care that the temperature of the cassette does not exceed 35 ° C.

Example 3-Fast format for detection of bacteria using non-proliferating nucleic acid probes with immobilized capture chemistry Medium copy number 4 from bacterial signal recognition particles (SRP). .5S RN
A fast, non-amplified diagnostic assay format based on A is described herein.
SRP is a ribonucleoprotein complex present in Escherichia coli at 400-1000 copies per cell (based on growth stage) (Batey).
Et al., Science 287: 1232-1239; (2000);
n and Pedersen, J .; Bacteriol. 176: 7148-71
54 (1994)). The 4.5S RNA or its homologs in bacteria vary considerably in size (~ 79 nucleotides (nt) in Mycoplasma pneumoniae, 315 nt in Bacillus brevis) and sequence. However, there is a conserved region of 22 nucleotides, which is targeted for probe development.

Bacterial lysis is achieved using heat and detergent, followed by rapid hybridization with a solution phase reporter probe conjugated to alkaline phosphatase (RP). The hybridization mixture is loaded into the wells of the gel cassette and subjected to high speed electrophoresis. The target RNA: RP complex is captured during electrophoresis by hybridization with a capture probe (CP) immobilized on an Acrydite ^™ chemical in polyacrylamide on polyacrylamide on a thin film (Kenny et al., Biotechniques). 25:
516-521 (1998); Rahenman et al., Nocleic Acid.
Research 27; 649-655 (1999)).

The membrane is washed and exposed to a chemiluminescent substance for detection. The entire assay, including the sample processing step, can be performed in about 60 minutes and is described below. col
Having a signal-to-noise value for i:

[0136]

[Chemical 1] .

These data show that y = 7.10 ^{−0.5 *} (x ^0.9497 ), R ² = 1.
A zero power curve (log: log) agreement is produced. This assay format provides simple hybridization chemistry, sensitivity to detection of bacteria using standard chemiluminescent agents, and rapid time to first results.

Example 4-Fast Assay for Bacterial Contamination Bacteria are spiked into platelet concentrates and concentrated by centrifugation. The sample is dissolved at elevated temperature in the presence of sodium dodecyl sulfate. The sample is hybridized with a high concentration of alkaline phosphatase conjugated reporter probe. The selection of probes was based on published sequence information (eg http://psyche.utthct.edu/dbs/srpdb/s).
rpdb. html).

Acrydite ^™ adhesion chemistry (US Pat. No. 5,932,711) allows immobilization of capture probes at high concentrations within a polyacrylamide gel matrix. The capture probe layer was supported on a thin film and inserted into an agarose-filled electrophoresis cassette 190, and the target molecule passed through the probe layer for rapid capture of the target and removal of excess unbound reporter probe. Subject to electrophoresis.

The capture gel holder 220, the capture membrane comb, was removed from the electrophoresis cassette 90 and placed in a tray (detection tray 78 in FIGS. 15A and 15B) designed to fit a standard 96-well microtiter plate footprint. Inserted. The chemiluminescent material was pipetted into these horizontal wells and the amount of luminescence was read with a luminometer at various times.

The signal to noise ratio (S / N) is calculated by reading the chemiluminescence data and comparing with the signal from a negative control matrix consisting of BSA and non-specific RNA. FIG. 23 shows the dose response to transcription of target in vitro spiked into this assay after centrifugation step. Figure 24 shows a similar dose response, but with E. coli spiked into sterile platelet concentrate. E. coli is used.

Table 1 shows a list of bacterial species detected with the current probe set using the thin gels described herein.

[0143]

[Table 1] Figure 25 shows the expected time to run 8 samples (4 samples per cassette and negative and positive controls, running 2 cassettes in parallel).

Example 5-Array Formation Each individual rectangle is incorporated into a matrix-forming gel solution with a unique probe. Rectangles, each provided with a specific probe or known probe set, were placed in a known pattern between two clean silanized glass plates separated by a spacer, and a sufficient acrylamide gel matrix was placed therein. Carry and form a thin gel around each rectangle and tie this rectangle to the sheet.

Example 6-Blot Electrophoresis Procedure for Rapid Hybridization Analysis of Bacterial Colonies Bacteria are grown on Petri plates. Blot onto a nylon membrane coated with agarose. Lyse the bacteria. A sandwich of nylon membrane and silk screen mesh is formed, the outer surface of each of the sandwiches is covered with Saran wrap, and a plate spacer is placed between the nylon membrane and silk screen mesh. Carry the gel film forming solution between the silk screen and the nylon mesh, and into any open space of the silk screen and the nylon mesh. Make the solution a gel.

Place the gel sandwich in a cassette filled with unsolidified agarose,
Then, solidify the agarose. An electric current is applied to the electrodes, thereby binding any analyte in the agarose on the nylon membrane proximal to the ligand bound to the thin gel matrix.

A label capable of binding the analyte-ligand complex is introduced and allowed to bind. The presence or absence of the label is detected by removing the thin gel matrix from the cassette and moving it to the reader for the label. Example 7-Detection of E. coli Contamination in Platelet Concentrate Samples were tested for E. coli in platelet concentrate. Prepare for detection of E. coli contamination. In this example, log phase E. coli was used to provide analysis of contaminated platelet samples. E. coli is added to the platelet sample. The nucleic acid probe used (probe 46 on capture gel 40) is directed towards the target (E. coli 4.5S RNA, also known as signal recognition particle RNA). The alkaline phosphatase-coupled detection probe is hybridized directly to the 4.5S RNA target. The adapter probe (block 154 in Figure 10) also hybridizes directly to the 4.5S RNA target at a position different from the position occupied by the detection probe. Some of the adapter probes are not complementary to the target,
It forms a single-stranded tail when hybridized to the target 4.5S RNA.
The single-stranded region of this adapter is complementary to the capture probe immobilized on an enhanced thin gel (see, eg, PCT / US00 / 08529, the teachings of which are incorporated herein by reference). That).

Concentration and lysis of the sample will be discussed with reference to block 150 in FIG. 1 (1.0) milliliter (ml) platelet sample was drawn and labeled in a tube (polypropylene screw cap storage vial for cryopreservation,
The volume was approximately 2 ml, Nalge). TE buffer (TE is 10 mMT
log phase E. coli in ris HCl (pH 8.3, 1 mM EDTA). c
Oli bacteria were spiked into platelet samples. The "negative" sample is
Accept only TE buffer. Other “positive” samples received 5 × 10 ⁵ colony forming units (cfu), 5 × 10 ⁶ cfu, and 5 × 10 ⁷ cfu. All samples were spun at 14,000 rpm for 60 seconds in a microcentrifuge (eg Eppendorf).

The supernatant was poured into biohazardous waste and the rim of the tube was blotted onto a clean paper towel. 100 μl of lysis buffer (lysis buffer is 100 mM sodium phosphate buffer (pH 7.0), 3 mM EDTA, 5% SDS, 0.
001% Phenol Red) is delivered to each tube. The pellet is suspended by vortex mixing or pipetting up and down with a micropipette.

Each tube was tightly capped, and as shown in block 152 of FIG. 10,
It was left to stand in a pre-warmed heating block at 135 ° C. for 5 minutes. After heating, transfer the tubes and place in a rack at room temperature for at least 90 seconds. In one embodiment,
Incubate the tube with the incubation unit 304 (
(For heating and cooling). Add 20 μl of 5 × DHB + AP to each tube using an Eppendorf repeater (5 × DHB + AP is 525 mM N
aCl, 0.5 mM MgCl ₂ , 0.05 mM ZnCl ₂ , 125 mM T
ris HCl (pH 8.3), 5% Ficoll 400, 0.05% wt
/ Vol xylene cyanol, 125 nanomolar (nM) alkaline phosphatase-conjugated oligodeoxynucleotide probe (3'-CTTCC GTCTA
CTGCG CACAC GG-alkaline phosphatase-5 ′) (SEQ ID NO: 19), 2.5 mM tricarboxylic acid aurine, 125 nM adapter oligonucleotide probe (3′-TCCGG GCCCT TGCAT AAGT).
G AGTCC AGGCC TTCCT TCGTC G-5 ') (SEQ ID NO: 20), which is 0.05% sodium azide). Manually shake each tube for a few minutes. Incubate the tubes at 52 ° C for 10 minutes to obtain 4.5S from bacteria.
The RNA is hybridized with a reporter probe and an adapter probe. 20 μl of hybridization mixture is loaded into sample well 174 in cassette 86 of FIG. 19B.

The electrode 100 is connected to a DC power source and a constant pressure of 50 V is applied for 10 minutes. During the run, the temperature rises, but should not rise above 35 degrees Celsius (C). If desired, the cassette can be placed on a surface cooled by a recirculating water bath to prevent overheating.

Referring to block 158 of FIG. 10, after moving the sample in the capture gel holder 66 toward the capture gel 40 for a period of time, the power is turned off. The capture gel holder 66 is moved from the expanding segment 98 in the flow channel 96 to another expanding segment 98 in the same electrophoresis cassette 86. Removal of the membrane from the sample electrophoresis cassette is facilitated by rocking the membrane back and forth before pulling it up.

The capture gel holder is moved from the expansion slot 98 of the first set of electrophoresis cassettes 86 and into the expansion slot, wash segment 98 of the electrophoresis cassette 86 of the second set. The teeth 70 of the capture gel holder 66 are placed in the buffer-filled slots created by the expansion slots 98.
The electrode 100 is connected to a DC power source and a constant pressure of 50V is applied for 5 minutes. again,
Be careful that the temperature of the cassette does not exceed 35 degrees.

As shown above, when discussing the method related to FIG. 10, the current is in the same direction during the second or wash period. By moving the capture gel holder 66 to the second enlarged segment 98, the prologation of the sample is
The capture gel holder 66 moves away from the capture gel 40, thus any sample on the capture gel holder 66 will pass uncaptured and no new sample will contact the capture gel 40.

After the above steps, detection of the nucleic acid target-alkaline phosphatase-bound probe complex captured on the capture gel 40 of the gel film, capture gel holder 66 is achieved. The capture gel holder 66 is removed from the electrophoresis cassette wash or second set of slots 98. The teeth 70 of the capture gel 40 are in a Tropix reagent (CDP star with Emerald II,
It is placed in the Tropix catalog MS100RY). The capture gel 40 is immersed in this solution and incubated for 15 minutes at room temperature with gentle shaking, as shown in FIG. 8 by reference numeral 170.

The capture gel holder 66 is then placed on the detection tray 178 as shown in FIGS. 15A and 15B. The capture gel holder 66 and the detection tray 178 are attached to a microplate luminometer 172 (eg, Walla Victor).
2, E. G. & G Walla, Turku, Finland). Read Relative Light Unit (RAU) and record both RAU and signal to noise ratio = (test sample membrane-empty well) / (negative control sample membrane-empty well). Figure 22 shows the results of three different experiments (Assay 1-3); the results are expressed as signal-to-noise ratio (S / N). The overall mean S / N ratio is also displayed along with its standard deviation. The samples tested were submitted to the indicated number of log phase E. E. coli bacteria (represented by the number of colony forming units (cfu) loaded per lane) are compared to spiked human platelet concentrates. The display “1.6E + 5” is an abbreviation for the value “1.6 × 10 ⁵ ”.

The above and other objects, features, and advantages of the present invention will be apparent from the following more detailed description of the preferred embodiments of the invention, which are illustrated in the accompanying drawings, wherein: Similar reference features refer to the same parts throughout the different drawings. These drawings need not be enlarged, but instead are highlighted in illustrating the principles of the invention.

[Brief description of drawings]

[Figure 1]   FIG. 1 is a top perspective view of an electrophoresis cassette.

[Fig. 2]   FIG. 2 is a top perspective view of an electrophoresis cassette with a cover.

[Figure 3]   FIG. 3 is a top view of the electrophoresis cassette.

FIG. 4A   FIG. 4A is a top perspective view of the evaporation cover.

FIG. 4B   FIG. 4B is a top view of the evaporation cover.

[Figure 5]   FIG. 5 is a front perspective view of the capture gel holder.

FIG. 6A   FIG. 6A is a front view of a capture gel holder.

FIG. 6B   FIG. 6B is a side view of the capture gel holder.

FIG. 7A   FIG. 7A is an enlarged front view of a portion of the capture gel holder of FIG. 6A.

FIG. 7B   7B is an enlarged front view of a portion of the capture gel holder of FIG. 6A.

FIG. 7C   7C is a cross-sectional view taken along line 7C-7C of FIG. 6A.

FIG. 7D   7D is a cross-sectional view taken along line 7D-7D of FIG. 6A.

[FIG. 7E]   7E is a cross-sectional view taken along line 7E-7E of FIG. 6A.

[FIG. 7F]   7F is a cross-sectional view taken along line 7F-7F of FIG. 6A.

FIG. 7G   7G is a cross-sectional view taken along line 7G-7G of FIG. 6A.

FIG. 8A is a top perspective view of a sample well forming comb for making sample wells in an electrophoresis cassette.

FIG. 8B   FIG. 8B is a top perspective view of the molding comb.

FIG. 8C   FIG. 8C is a top perspective view of an alternative molding comb.

FIG. 9A is a schematic diagram of an exemplary method of determining whether bacteria are present in a biological sample.

FIG. 9B is a schematic diagram of an exemplary method of determining whether a second bacterium is present in a biological sample.

FIG. 9C shows the RNA sequences of SEQ ID NOs: 1-17 from a panel of target organisms, along with the complement of probe sequences. The line surrounded by a thin box indicates the DNA sequence complementary to the probe. The white dotted line is below the sequence corresponding to the capture probe linked to the thin gel membrane. The white solid line is below the sequence corresponding to the reporter probe.

FIG. 10 is a schematic diagram of a method for rapid assay of an analyte in a sample.

FIG. 11A FIG. 11A shows a front perspective view of a capture gel reinforced with a non-conductive mesh.

11B is a cross-sectional view of the capture gel taken along line 11B-11B of FIG. 11A.

FIG. 12A shows a side view of an alternative embodiment of a capture gel with meshed encapsulated glass fibers.

12B is a cross-sectional view of the capture gel taken along line 12B-12B of FIG. 12A.

[Fig. 13]   FIG. 13 is a side perspective view of the electrophoretic device with the sides removed.

FIG. 14 is a side perspective view of the electrophoretic device and the incubator unit thereon.

FIG. 15A is a top view of a tray for housing a set of capture gel holders for use in a detection device (eg, luminescence reader).

15B is a cross-sectional view of the tray taken along line 15B-15B of FIG. 15A.

FIG. 16 is a schematic representation of an embodiment of electrophoretic cassettes and detection conditions in which target detection is achieved by an enzyme-stimulated chemiluminescence reaction.

FIG. 17   FIG. 17 is an outline of the purification method.

FIG. 18A   FIG. 18A is a front view of a capture gel holder.

FIG. 18B   18B is a perspective view of the capture gel holder of FIG. 18A.

FIG. 19A   FIG. 19A is a top view of an alternative embodiment of an electrophoresis cassette.

19B is a cross-sectional view of the electrophoresis cassette taken along line 19B-19B of FIG. 19A.

FIG. 19C   FIG. 19C is a side perspective view of the electrophoresis cassette.

FIG. 20 is a side perspective view of a comb for making sample wells in an electrophoresis cassette.

FIG. 21A shows a top view of an alternative embodiment of an electrophoretic parallel channel device.

FIG. 21B   21B is a cross-sectional view of the electrophoresis cassette of FIG. 21A.

FIG. 21C]   FIG. 21C shows a capture gel holder with a thin layer gel or a reinforced thin layer gel.

FIG. 22   FIG. 22 shows a graph of the results obtained using the disclosed assay.

FIG. 23 is a graph showing the dose response to in vitro target transcripts added to the assay after the centrifugation step.

FIG. 24. E. coli added to sterile platelet concentrate. 24 is a graph showing a similar dose response of FIG. 23 using E. coli.

FIG. 25 shows an estimate of the time to electrophorese up to 8 samples in the assay method described herein.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 27/26 315A (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI) , FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE) , SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ , LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Abrams, Ezraes. United States Massachusetts 02465, Newton, Colbert Rod 4 (72) Inventor Kiefer-Higgins, Stephen Gee. United States Massachusetts 02115, Boston, Park Drive No. 6 117 (72) Inventor Zan, Tian Hong United States Massachusetts 01721, Ashland, Running Brook Circle 22 (72) Inventor Ecock, Grampie. United States Massachusetts 01752, Marlborough, Flanders Road 5 (72) Inventor Web, Michael Jay. United States Massachusetts 01827, Dance Table, French Court 39 (72) Inventor McDowell, Christopher United States Massachusetts 02767, Raynham, Orchard Street 519 F Term (reference) 2G045 BB01 BB10 BB50 BB51 DA13 FB02 FB05 4B029 AA07 A20 A18B4A29A18B27AA27B27AA27 QA20 QQ42 QR08 QR42 QR56 QS16 QS25 QS34 QS39 QX02

Claims (66)

[Claims] 1. An apparatus for capturing an analyte, comprising: an electrophoresis cassette, the following: a pair of electrode channels, and a barrier interposed between the electrode channels. A base having an expansion slot, the barrier having at least one migration channel extending between the electrode channels, the expansion slot binding to and opening to the migration channel. An electrophoretic cassette comprising: a base; a first electrode extending into a first electrode channel; a second electrode extending into a second electrode channel; and a capture gel holder receivable in the expansion slot, the capture gel comprising: A capture gel holder, wherein the holder has an opening aligned with the migration channel. 2. The apparatus of claim 1, wherein the barrier is coupled to the migration channel and is open to the migration channel for receiving the capture gel holder. A device having a slot. 3. The apparatus according to claim 2, further comprising an evaporation cover that covers the electrophoresis cassette, the evaporation cover being at least one opening for the capture gel holder and for venting gas. An apparatus having at least one opening in. 4. The apparatus according to claim 3, wherein the electrophoresis cassette comprises at least one wash well. 5. The apparatus of claim 3, wherein at least one of the electrodes comprises a pair of terminals extending through the evaporation cover and flush with the top of the evaporation cover. apparatus. 6. The apparatus of claim 3, wherein the capture gel holder comprises a plurality of teeth, each tooth having an opening for receiving a non-conductive polymer mesh, and the teeth. An apparatus having a polar device so that the teeth fit in an enlarged slot of the electrophoresis cassette in a specific manner. 7. The device of claim 4, wherein the capture gel holder comprises a plurality of teeth, each tooth having an opening for receiving a non-conductive polymer mesh, and the teeth. An apparatus having a polar device so that the teeth fit through at least one opening in the evaporative cover. 8. An apparatus for capturing an analyte, comprising: an electrophoresis cassette, the following: a pair of electrode channels, and a barrier interposed between the electrode channels. A base having an expansion slot, the barrier having at least one migration channel extending between the electrode channels, the expansion slot binding to and opening to the migration channel; An electrophoretic cassette comprising: a base; a first electrode extending into a first electrode channel; a second electrode extending into a second electrode channel; a capture gel holder receivable in the expansion slot, the capture gel A holder having an opening aligned with the migration channel; a thin gel supported on an opening of a capture gel holder, the thin gel comprising a gel Helix and a covalently bound ligand of the gel matrix, thin layer gel. 9. The device of claim 8, wherein the lamina gel further comprises a non-conductive polymer mesh for interlocking with the gel matrix. 10. The apparatus of claim 9, wherein the barrier is coupled to the migration channel for receiving the capture gel holder and is open into the migration channel. A device having a slot. 11. The apparatus according to claim 10, further comprising an evaporation cover covering the electrophoresis cassette, the evaporation cover being at least one opening for the capture gel holder and for venting gas. Having at least one opening of
apparatus. 12. The apparatus according to claim 11, wherein the electrophoresis cassette comprises at least one wash well. 13. The apparatus of claim 11, wherein at least one of the electrodes comprises a pair of terminals extending through the evaporation cover and flush with the top of the evaporation cover. apparatus. 14. The device of claim 11, wherein the capture gel holder comprises a plurality of teeth, each tooth having an opening for receiving a non-conductive polymer mesh, and the teeth. An apparatus having a polar device so that the teeth fit in an enlarged slot of the electrophoresis cassette in a specific manner. 15. The apparatus of claim 14, wherein the capture gel holder further comprises a detection surface. 16. The apparatus of claim 11, wherein the capture gel holder comprises a plurality of teeth, each tooth having an opening for receiving a non-conductive polymer mesh, and the teeth. An apparatus having a polar device so that the teeth fit through at least one opening in the evaporative cover. 17. A capture gel holder comprising: a handle; a plurality of teeth projecting from the handle, at least one of which has a hole therethrough; a gel matrix and the gel matrix; A capture gel holder comprising: a ligand covalently bound to the gel matrix covering the wells. 18. The capture gel holder of claim 17, wherein the at least one tooth has a conforming shape adapted to conform to the electrophoresis cassette only in a particular orientation. holder. 19. The capture gel holder of claim 18, wherein each of the teeth has a concave central region around the hole and a flange over the hole, wherein the concave region. And the flange is for facilitating the release of gas, the capture gel holder. 20. The capture gel holder according to claim 19, further comprising:
A capture gel holder that covers each hole in the tooth and comprises a non-conductive polymeric material to support the gel matrix and ligand. 21. The capture gel holder of claim 20, wherein the conforming shape is adapted to conform to the electrophoresis cassette only in a particular orientation.
Capture gel holder, including each tooth with curved and flat edges. 22. The capture gel holder according to claim 21, wherein the conforming shape has a protrusion adapted to pass only a specific slot in the evaporation cover of the electrophoresis cassette. Capture gel holder, including teeth. 23. The device of claim 21, wherein the capture gel holder further comprises a detection surface. 24. The apparatus according to claim 20, wherein said conformable shape comprises a tooth having a protrusion adapted to pass through only a specific slot in the evaporation cover of the electrophoresis cassette. Including, device. 25. A thin gel for electrophoretic capture of an analyte, comprising: a gel matrix and a ligand covalently attached to the gel matrix. 26. The thin gel of claim 25, wherein the gel matrix has sufficient tensile strength to allow removal from the electrophoretic device. 27. The thin layer gel of claim 25, further comprising a non-conductive polymeric material. 28. The lamellar gel of claim 27, wherein the non-conductive polymeric material is selected from the group consisting of mesh, mats, fabrics, and felts. 29. The lamellar gel of claim 27, wherein the non-conductive polymeric material is a polymer capable of cross-linking the gel matrix. 30. A device for capturing an analyte, the device comprising:   A gel matrix and a ligand covalently bound to the gel matrix, A device. 31. The device of claim 30, further comprising a non-conducting polymeric material to support the gel matrix and ligand. 32. The device of claim 31, further comprising a capture gel holder having a plurality of openings for receiving the non-conductive polymeric material. 33. The apparatus of claim 32, further comprising an electrophoresis cassette for receiving a gel for electrophoresis and capture of analytes, the electrophoresis cassette comprising: A base having electrode channels, a barrier inserted between the electrode channels, and an expansion slot, the barrier having at least one migration channel extending between the electrode channels; A base bound to and open to the migration channel for receiving the capture gel holder; a first electrode extending to a first electrode channel; a first electrode extending to a second electrode channel; A device comprising two electrodes; 34. The device of claim 33, wherein the barrier is coupled to the migration channel for receiving the capture gel holder and is open to the migration channel. Having a device. 35. A method for capturing a target molecule contained in a sample, comprising the steps of: non-conducting polymeric material having a gel matrix containing a covalently bound ligand specific for the target molecule. Providing the sample through a non-conductive polymeric material having the gel matrix, the target molecules being captured by capture probes of the gel matrix, and the rest of the sample passing through the non-conductive polymeric material. The method comprising the steps of: 36. The method of claim 35, wherein the target molecule is detectably labeled prior to passage of the sample through the gel matrix.
Method. 37. The method for capturing a target molecule according to claim 29, further comprising the following steps: providing an electrophoresis cassette having an electrophoresis channel extending between a pair of electrode channels each having an electrode; Providing an electrophoretic matrix in an electrophoretic cassette and forming a sample-well for receiving a sample in the electrophoretic channel; a non-conducting polymer having a gel matrix containing covalently bound ligands carried by a capture gel holder Inserting material into the migration channel by placing the capture gel holder in the expansion slot, the expansion slot binding to the migration channel and opening into the migration channel. The step of inserting the sample into the sample well together with the target molecule. ; Through voltage in the electrophoretic cassette, comprising the steps, for electrophoresis the samples toward from the sample well to the non-conductive polymeric material in said channel. 38. The method of claim 37, further comprising the steps of: removing the capture gel holder from the electrophoresis cassette; and a reader for detecting a probe associated with an analyte. Placing a capture gel holder. 39. The electrophoresis cassette having a second expansion slot for each migration channel for receiving the capture gel holder.
The method described in. 40. The method of claim 37, further comprising: removing the capture gel holder from the electrophoresis cassette; and separating the capture gel holder between the capture probe and the target molecule. Subjecting the cells to conditions sufficient to break the bond of the. 41. The electrophoresis cassette having a second expansion slot for each migration channel for receiving the capture gel holder.
The method described in. 42. A method of detecting a target molecule comprising the steps of: comprising a capture gel holder having a non-conducting polymeric material having a gel matrix containing a covalently bound ligand; a migration channel and a pair of expansions. Providing an electrophoretic cassette having a slot, the electrophoretic channel extending between a pair of electrodes and a sample well for receiving a sample in the electrophoretic channel, the pair of expanding slots being ,
Binding to and opening to the electrophoretic channel; loading the sample with target molecules into the sample well; placing the capture gel holder in a pair of electrophoretic channels Inserting into the expansion slot; passing a voltage through the electrophoresis cassette to migrate the sample from the sample well in the migration channel toward the non-conducting polymeric material; removing the capture gel holder from the electrophoresis cassette. Removing; and placing the capture gel holder in a reader to detect the probe associated with the analyte. 43. The method of claim 42, further comprising the steps of: preparing a sample containing a reporter probe attached to the target molecule; interrupting the voltage in the electrophoresis cassette. Moving the capture gel holder to a washing station; inserting the capture gel holder into another expansion slot in the migration channel; and passing a voltage across the electrophoretic matrix in the electrophoresis cassette in the channel. Migrating the sample from the sample well away from the capture gel holder and the non-conducting polymeric material. 44. A method for performing an analyte ligand binding assay, the method comprising the steps of: a) comprising an electrophoresis cassette, the cassette comprising: a pair of electrodes. A base unit having a channel, a barrier inserted between the electrode channels; an expansion slot, the barrier having at least one migration channel extending between the electrode channels, the expansion slot A base unit coupled to and opening to the migration channel to receive a capture gel holder; a first unit extending to the first electrode channel; a second unit extending to the second electrode channel A second electrode; a sample-well forming comb removably mounted in the migration channel; a gel matrix and the gel matrix A thin layer gel bound and having a ligand located in the migration channel; b) filling the device with a gel for electrophoresis; c) curing the gel; d) Removing the comb, thereby creating a sample well; e) placing the sample in the sample well; f) providing an electromotive force; and g) moving the sample through the thin layer gel by the electromotive force. The method comprising: 45. The method of claim 44, further comprising providing a detection probe in the sample well. 46. The method of claim 44, further comprising the step of: g
) Removing the thin gel from the device, and h) detecting the presence or absence of the detection probe. 47. The method of claim 46, further comprising the step of: washing the lamina gel between steps g) and h). 48. A device for capturing a target molecule comprising: a housing comprising: a first electrode channel, a second electrode channel, and between the first electrode channel and the second electrode channel. A base having an inserted barrier and an enlarged slot,
The barrier has at least one migration channel extending between the first electrode channel and the second electrode channel, and the expansion slot is coupled to the migration channel,
A base opening into the migration channel; a slot, the capture gel holder having openings aligned with the migration channel; and a gel supported by the openings in the capture gel holder, the thin gel A gel having a gel matrix and a ligand covalently attached to the gel matrix,
A housing having a housing. 49. The device of claim 48, further comprising a first electrode extending into the first electrode channel and a second electrode extending into the second electrode channel. 50. The device of claim 48, wherein the gel further comprises a non-conductive polymer mesh for connection with the gel matrix. 51. The device of claim 50, wherein the barrier is coupled to the migration channel for receiving the capture gel holder and has a second expansion slot open to the binding channel. Having a device. 52. The apparatus of claim 51, further comprising an evaporation cover for covering the electrophoresis cassette, the evaporation cover having at least one opening for the capture gel holder and a gas barrier. A device having at least one opening for venting. 53. The method of claim 52, wherein the electrophoresis cassette comprises at least one wash well. 54. The device of claim 52, wherein at least one of said electrodes has a pair of terminals extending through said evaporative cover and flush with the top of said evaporative cover. . 55. The apparatus according to claim 52, wherein the capture gel holder has a plurality of teeth, the teeth adapted in a particular manner into the expansion slots of the electrophoresis cassette. An apparatus having a polar device as described above. 56. A capture gel holder comprising: a handle; a plurality of teeth projecting from the handle, at least one of the teeth having a hole therethrough; and covering the hole. A capture gel holder comprising: the gel matrix; 57. A capture gel holder according to claim 56, wherein at least one tooth is adapted to allow an arrangement adapted to fit the electrophoresis cassette only in a particular orientation. Capture gel holder with a curved shape. 58. The capture gel holder of claim 57, wherein each tooth has a concave central region around the hole and a flange over the hole, wherein the concave region. And the flange may facilitate release of gas, a capture gel holder. 59. The capture gel holder of claim 58, further comprising:
A capture gel holder comprising a non-conducting polymeric material to cover each hole of the tooth and to support the gel matrix and ligand. 60. The capture gel holder of claim 59, wherein the conforming shape is adapted to fit the electrophoresis cassette only in a particular orientation.
Capture gel holder, including each tooth with curved and flat edges. 61. A capture gel holder according to claim 60, wherein the conforming shape comprises teeth having protrusions adapted to pass only through specific slots in the evaporation cover of the electrophoresis cassette. , Capture gel holder. 62. The apparatus of claim 60, wherein the capture gel holder further comprises a detection surface. 63. The apparatus according to claim 59, wherein the conforming shape comprises teeth having protrusions adapted to pass only through specific slots in the evaporation cover of the electrophoresis cassette. . 64. A gel for capturing an analyte by electrophoresis, comprising:
A gel comprising a gel matrix and a ligand covalently attached to the gel matrix. 65. The gel of claim 64, wherein the gel matrix has sufficient tensile strength to allow removal from an electrophoretic device. 66. The gel of claim 64, further comprising a non-conductive polymeric material.