ES2356777T3 - BIOCHEMISTRY BASED ON DROPS. - Google Patents

Abstract

A method for providing a drop in contact with a surface of one or more magnetically sensitive beads (1102, 1207, 1305) comprised in the drop in a microactor of drops, with a reduced concentration of the substance, the process comprising: (a) providing a drop microactor comprising said surface of one or more magnetically sensitive beads in contact with the drop (1101, 1206), the drop comprising a starting concentration and a starting amount of a substance (1103, 1208, 1306) and having a starting volume; (b) perform one or more drop operations to join a wash drop (1106, 1205) with the drop (1101, 1206) provided in step (a) to give a combined drop (1307); and (c) immobilize the one or more magnetically sensitive beads by exposing said one or more magnetically sensitive beads to a magnetic field; characterized in that while said one or more magnetically sensitive beads are thus immobilized, said method further includes the step of: (d) performing one or more operations of electrohumidification-mediated drops to divide the combined drop to give a set of drops comprising: - (i) a drop (1108, 1212) in contact with said surface of one or more magnetically sensitive beads having a decreased concentration of the substance with respect to the starting concentration; and - (ii) a drop (1110, 1214) that is separated from said surface from one or more magnetically sensitive beads.

Description

1 Field of the Invention

The invention relates to a drop microactor and to systems, apparatus and methods that employ the drop microactor to execute various protocols using separate drops. The invention is useful, for example, in the execution of protocols for performing biochemical and / or biological tests. The invention also relates to systems and methods for executing protocols based on drops.

2 Background of the Invention

The ability to quickly perform biochemical and other tests is critical in a wide variety of fields. For example, a rapid and accurate diagnosis of infectious disease is crucial both for the effective management of the disease in individual subjects and for the improvement of public health problems of pathogens with multi-drug resistance and out-of-hospital infections.

Current PCR-based DNA amplification procedures experience several drawbacks including the high cost of reagents, labor intensity and susceptibility to cross contamination. In addition, compared to culture, PCR assays are less capable of simultaneously testing multiple species, virulence factors and drug resistance markers. Frequently, they lack sensitivity and cost-effective quantification of the pathogen. There is a need in the art for improved devices for the detection of nucleic acids that overcome these limitations while also miniaturizing and automating the technique, so that these tests could potentially be applied at the point of sample collection with minimal training.

Nucleic acid sequencing is becoming increasingly common in a variety of fields, such as complete genome sequencing, diagnosis, pharmacogenomics and forensic medicine. However, the field of sequencing has been hampered by the expensive nature of sequencing machines. The development of economic high performance test systems is critically important for the generalization of the genetic test and the many advantages that are associated with it. Therefore, there is a need for new technological platforms that allow to quickly and reliably sequence nucleic acids at a reasonable cost. The invention described herein provides an economical drop-based sequencing system.

Immunoassays are widely used for clinical diagnosis and constitute a market of more than 3 billion dollars in the United States alone. Immunoassays are among the most sensitive and specific procedures that are routinely used in a clinical laboratory. Immunoassays make use of the high affinity and specificity in the binding between an antigen and its homologous antibody to detect and quantify the antigen in a sample matrix. Heterogeneous immunoassays, such as ELISA (enzyme-linked immunosorbent assay), are among the most sensitive and specific clinical analysis procedures, and have been widely used for the identification of a broad class of antigens and antibodies. For example, immunoassays are performed, among other things, for the identification of cardiac markers, tumor markers, drugs, hormones and infectious diseases.

The lower sample consumption, faster analysis and complete automation are three highly desirable characteristics that require continuous improvement of any clinical analyzer. Although state-of-the-art laboratory immunoassay analyzers offer good automation and performance, they require a significant amount of sample per assay (including unusable volumes) and prolonged analysis times. The long test times and the large size of these analyzers makes them impractical for use in a sample collection point environment.

In addition, there is considerable variability in immunoassay performance, largely attributed to the fact that the techniques depend on the operator, resulting in it being difficult to compare results from one study to another, and even within the same study if more than one use is used. laboratory. A fully automatic and integrated analyzer that eliminates operator dependence and normalizes the results for immune control tests would significantly improve the interpretation of test results.

Although significant advances have been made in the automation of immunoassays, these analyzers are prohibitively expensive and a low-performance research environment cannot afford them. Low-performance systems with automatic plate washers, incubators and integrated optics still require an expert technician to perform several key stages in an immunoassay, such as preparing microtiter plates with antibodies and loading samples onto the plates. This results in human errors due to repeated manual intervention, and is a very important source of inter-trial and intra-trial variation.

There is also a need for tests at the point of sample collection in a variety of fields, such as detection fields related to medicine, the environment and bioterrorism. As an example, the trial at the point of sample collection (PRM) for clinical blood tests has improved, but remains a key challenge for modern medical care. Ideally, the PRM trial would allow the clinician to diagnose and apply life-saving technologies in real time, avoiding the need for large laboratory facilities. There is still a need in the art for a laboratory on a chip that allows simultaneous control of gases, metabolites, electrolytes, enzymes, DNA, protein and blood cells in low sample volumes in the PRM.

Microfluidic fluid control is an essential requirement for a successful chip laboratory. Microfluidic systems can be broadly grouped into architectures based on continuous flow and discrete flow. As the name suggests, continuous flow systems depend on the continuous flow of liquids in channels while discrete flow systems use liquid drops within channels

or in an architecture without channels. A common limitation of continuous flow systems is that the liquid transport is physically confined to permanently fixed channels. The transport mechanisms used are usually pressure driven by external pumps or electrokinetic driven by high voltages. These strategies involve complex channeling and require large support systems in the form of external valves or power supplies. These restrictions make it difficult to achieve a high degree of functional integration and control in conventional continuous flow systems, particularly in the realization of a handheld device at the sample collection point. There is still a need in the art for a sample collection point test system that makes use of drop manipulations and, especially, a system that can perform multiple tests or multiple types of tests in a single microchip.

3 Brief Description of the Invention

One aspect of the present invention relates to a drop based wash. Another aspect of the present invention relates to a surface modification and a washing based on drops. Therefore, in accordance with the present invention, there is provided a method for providing a drop in contact with the surface of one or more magnetically sensitive beads comprised in the drop in a drop microactor, the drop comprising a starting concentration and a starting amount of a substance and having a starting volume, with a reduced concentration of the substance, the process comprising:

(to)

 providing a droplet microactor comprising the surface in contact with the drop; Y

(b)

perform one or more operations of drops to join a drop of washing with the drop provided in step (a), to give a combined drop;

characterized in that said procedure also includes the steps of:

(C)

 immobilize magnetically sensitive beads by exposing magnetically sensitive beads to a magnetic field; Y

(d)

Perform one or more operations of electrohydrated mediated drops to divide the combined drop to give a set of drops comprising:

(i)

 a drop in contact with the surface having a decreased concentration of the substance with respect to the starting concentration; Y

(ii)

 a drop that is separated from the surface.

In one embodiment, the method further comprises releasing or resuspending one or more of the beads after step (d). In an alternative embodiment, step (c) comprises placing the drop comprising the magnetically sensitive beads in proximity to a means to achieve a magnetic field in proximity to one or more electrodes, in which the intensity and proximity of the magnetic field are sufficient to magnetically immobilize sensitive pearls during the execution of a washing protocol.

In a further alternative embodiment, step (d) produces a set of drops comprising:

(to)

 a drop comprising substantially all the beads and having a decreased concentration of the substance with respect to the starting concentration; Y

(b)

 a drop that lacks substantially pearls.

In further preferred embodiments, the drop of step (d) (i) comprises more than 99%, more preferably more than 99.999%, of the beads present in the combined drop.

4 Definitions

As used herein, the following terms have the indicated meanings.

The term "activate", in reference to one or more electrodes, refers to effecting a change in the electrical state of one or more electrodes that results in a droplet operation.

"Affinity" means the specific or nonspecific intramolecular attraction of a molecule by another molecule or by a substrate, such as the attraction of an antibody by its corresponding antigen or hapten.

"Analyte" means a target substance for detection that may be present in a sample. Illustrative examples include antigenic substances, haptens, antibodies, proteins, peptides, amino acids, nucleotides, nucleic acids, drugs, ions, salts, small molecules, cells.

The "antibody" means a polypeptide that has an affinity for an epitope or hapten. An antibody can be a polyclonal antibody, a monoclonal antibody, an antibody fragment and / or a genetically engineered molecule capable of binding to the corresponding element of a specific binding pair. Antibodies can be labeled or otherwise conjugated with molecules that facilitate direct or indirect detection of and / or quantification of the antibody.

The term "pearl", with respect to pearls in a microactor of drops, means any pearl or particle capable of interacting with a drop in or near a microactor of drops. The beads can have any of a wide variety of shapes, such as spherical, spherical in general, egg-shaped, disk-shaped, cubic or other three-dimensional shapes. For example, the pearl may be able to be transported in a drop in a microactor of drops; configured with respect to a droplet microactor in a way that allows a drop in the droplet microactor to contact the bead, in the droplet microactor and / or out of the droplet microactor. Pearls can be made using a wide variety of materials, including, for example, resins and polymers. The beads can be of any suitable size, including, for example, microbeads, microparticles, nanobeads and nanoparticles. In some cases, the pearls are magnetically sensitive; In other cases, pearls are not significantly magnetically sensitive. For magnetically sensitive pearls, the magnetically sensitive material may constitute substantially the entire pearl or only a component of a pearl. The rest of the pearl may include, among other things, polymeric material, coatings and residues that allow the binding of a test reagent. Examples of suitable magnetically sensitive beads are described in US Patent Publication No. 2005-0260686, "Multiplex flow assays preferably with magnetic particles as solid phase," published November 24, 2005, the full description of which is incorporated in the present document by reference regarding its teachings in relation to magnetically sensitive materials and pearls.

The term "dNTP" means a deoxynucleotide triphosphate, in which the nucleotide is any nucleotide, such as A, T, C, G or U. The term "ddNTP" refers to a dideoxynucleotide triphosphate, in which the nucleotide is any nucleotide, such as A, T, C, G or U. It will be appreciated that unless specifically indicated otherwise, a ddNTP can be replaced by dNTP and vice versa.

The term "drop" means a volume of liquid in a microactor that is at least partially limited by loading fluid. For example, a drop may be completely surrounded by loading fluid or it may be limited by loading fluid and one or more surfaces of the drop microactor. Drops can take a wide variety of forms; Non-limiting examples generally include disc-shaped, ingot-shaped, truncated sphere, ellipsoid, spherical, partially compressed, hemispherical, ovoid, cylindrical sphere and various shapes formed during droplet operations, such as joining or excision, or formed as a result of the contact of said forms with one or more surfaces of a microactor of drops.

The expression "drop operation" means any manipulation of a drop in a microactor of drops. A drop operation may include, for example: loading a drop into the drop microactor; dispense one or more drops from a source drop; split, separate or divide a drop into two or more drops; transport a drop from one location to another in any direction; join or combine two or more drops in a single drop; dilute a drop; mix a drop; shake a drop; deform a drop; hold a drop in position; incubate a drop; heat a drop; vaporize a drop; cool a drop; get rid of a drop; transport a drop out of a drop microactor; other drop operations described herein; and / or any combination of the above. The terms "join", "that unites", "combine", "that combines" and the like are used to describe the creation of a drop from two or more drops. It should be understood that when such a term is used in reference to two or more drops, any combination of drops operations sufficient to result in the combination of the two or more drops in one drop may be used. For example, "joining of the drop A with the drop B" can be achieved by transporting a drop A in contact with a stationary drop B, transporting a drop B in contact with a stationary drop A, or transporting the drops A and B in contact with each other. The terms "split", "separate" and "divide" are not intended to imply any particular result with respect to the size of the resulting drops (ie, the size of the resulting drops may be the same or different) or the number of resulting drops ( the number of resulting drops can be 2, 3, 4, 5 or more). The term "mix" means drop operations that result in a more homogeneous distribution of one or more components within a drop. Examples of “load” droplet operations include microdialysis loading, pressure-assisted loading, robotic loading, passive loading and pipette loading.

The term "electronically coupled" is used herein to indicate an electrical or data relationship between two or more components or elements. As such, the fact that a first component is said to be electronically coupled to a second component is not intended to exclude the possibility that additional components may be present between and / or operatively associated or engaged with the first and second components. In addition, electrically coupled components may include wireless intermediate components in some embodiments.

The term "magnetically sensitive" means sensitive to a magnetic field. Examples of magnetically sensitive materials include paramagnetic materials, ferromagnetic materials, ferrimagnetic materials and metamagnetic materials. Examples of suitable paramagnetic materials include iron, nickel and cobalt, as well as metal oxides, such as Fe3O4, BaFe12O19, CoO, NiO, Mn2O3, Cr2O3 and CoMnP.

The term "protocol" means a series of steps that include, but are not limited to, operations of drops in one or more microactivators of drops.

The term "surface," in relation to the immobilization of a molecule, such as an antibody or analyte, on the surface, means any surface on which the molecule can be immobilized while retaining the ability to interact with drops in a microaction. of drops. For example, the surface may be a surface in the microactor of drops, such as a surface in the upper plate or lower plate of the microactor of drops; a surface extending from the top plate or bottom plate of the microaction actuator; a surface on a physical object located in the droplet microactor in a way that allows it to interact with droplets in the droplet microactor; and / or a pearl located in the microactor of drops, for example, in a drop and / or in a microactor of drops, but external to the drop.

The term "wash", with respect to washing a surface, means reducing the amount of a

or more substances in contact with the surface or exposed to the surface of a drop in contact with the surface. The reduction of the amount of the substance can be partially, substantially complete or even complete. The substance may be any of a wide variety of substances; Examples include target substances for further analysis and unwanted substances, such as components of a sample, contaminants and / or excess reagents. The surface may be, for example, a surface of a microactor of drops or a surface of a pearl in a microactor of drops. In some embodiments, a washing operation begins with a starting drop in contact with a surface, in which the drop includes an initial total amount of a substance. The washing operation can be developed using a variety of drop operations. The washing operation can produce a drop in contact with the surface, in which the drop has a total amount of the substance that is less than the initial amount of the substance. In another embodiment, the operation of drops may produce the surface in the absence of a drop, in which the total amount of the substance in contact with the surface or otherwise exposed to the surface is less than the initial amount of the substance in contact with the surface or exposed to the surface in the drop of departure. Other embodiments are described elsewhere herein, and others will be immediately apparent in view of the present disclosure.

When reference is made to a component given herein, such as a layer, region or substrate, as arranged or formed "on" another component, that given component may be directly on the other component or, as alternatively, may also be present intermediate components (for example, one or more coatings, layers, interlayers, electrodes or contacts). It will also be understood that the expressions "arranged on" and "formed on" are used interchangeably to describe how a given compound is positioned or placed in relation to another component. Therefore, the terms "arranged on" and "formed on" are not intended to introduce any limitations in relation to particular procedures for transport, deposition or manufacturing of material.

When it is described that a liquid in any form (for example, a drop or a continuous body, either moving or stationary) is "on," "on" or "above" an electrode, series, matrix or surface , said liquid could be in direct contact with the electrode / series / matrix / surface or it could be in contact with one or more layers or films that are interposed between the liquid and the electrode / series / matrix / surface.

When it is described that a drop is "on" or "loaded on" a drop microactor, it should be understood that the drop is disposed on the drop microactor so as to facilitate the use of the drop microactor to perform drop droplet operations. , the drop is arranged on the drop microactor so as to facilitate the detection of a property of or a drop signal, and / or the drop has undergone a drop operation on the drop microaction.

5 Brief Description of the Drawings

Figures 1 and 2 are illustrations showing reaction steps and drop operations of an illustrative embodiment in accordance with the present invention;

Figure 3 is a perspective view of a drop microactor for use in performing immunoassays in accordance with an embodiment of the present invention;

Figure 4 is an illustration showing steps for performing a drop-based sandwich affinity-based assay performed on a microaction dropper according to an embodiment of the present invention;

Figure 5 is an illustration showing steps to perform a competitive affinity-based assay performed on a microaction dropper according to an embodiment of the present invention;

Figures 6-8 are illustrations showing steps to immobilize and release magnetically sensitive beads using a magnetic field according to various embodiments of the present invention;

Figure 9 is an illustration of micro-actuator drop systems in accordance with an embodiment of the present invention;

Figures 10A and 10B are illustrations of a portable handheld analyzer according to an embodiment of the present invention;

6 Detailed Description of the Invention

The invention provides methods, devices and systems for executing one or more biochemical tests based on drops. For example, the invention provides methods, devices and systems for amplifying nucleic acids, analyzing nucleic acid sequences, performing affinity-based assays and / or analyzing body fluid components.

In certain embodiments, a system protocol may involve one or more of the following steps in any order that achieves the purpose of detecting the invention: extracting a sample from a subject; process the sample for loading in a drop microactor; load the sample into the drop microactor; Dispense one or more drops of the sample from the sample for transport in the drop microactor; load one or more reagents in the drop microactor; Dispense one or more drops of reagent for transport in the drop microactor; transporting one or more drops of reagent and / or one or more drops of sample to put the one or more drops of reagent in contact with the one or more drops of sample, thereby achieving the interaction of the reagent with the sample; detect an effect of the interaction of the reagent with the sample; provide a result that notifies the user of the results of the detection stage. Examples of biochemical protocols for use with a droplet microactor of the invention in the subsequent sections are analyzed.

6.1 Nucleic Acid Amplification

The invention provides methods, devices and systems for the amplification of nucleic acids in drops in a microactor of drops. A large number of copies of one or more nucleic acid molecules can be generated in a single drop from a small number of copies or even a single copy present in the drop. The methods of the invention generally involve combining the reagents necessary to form a drop ready for amplification and thermocycling of the drop at temperatures sufficient to result in the amplification of a target nucleic acid, for example, by chain reaction of the polymerase (PCR). In some embodiments, a drop that includes the amplified target nucleic acid can then be transported to a subsequent procedure, such as a detection procedure, a procedure for further manipulation of the target nucleic acid and / or a sequencing procedure. The amplification devices may include a microactor of drops and components sufficient to perform operations of drops that affect the methods of the invention when the microactor of drops is loaded with appropriate reagents. The systems of the invention may include the drop microactor plus system components designed to facilitate software control of the operation of the drop microactor to execute protocols of the invention.

In one embodiment, the invention provides a drop microactor and / or a system configured and programmed to effect the amplification of a sample in a drop ready for amplification, followed by the capture of the amplified nucleic acid. The amplified nucleic acid can be treated to denature it in single stranded nucleic acid before or after it is contacted with magnetically sensitive beads to allow the single stranded nucleic acid to bind to magnetically sensitive beads. Binding, for example, can be achieved using a biotin-streptavidin system, for example, in which the single stranded nucleic acid is biotinylated and the surface (e.g., beads or surface of a microaction dropper) is coated with covalently bound streptavidin. to the same. Amplification reagents can be washed away using a wash protocol. Various other procedures, devices, systems and other aspects of the invention will be apparent from the subsequent discussion.

6.1.1 Samples and Sample Preparation

The amplification procedures of the invention make use of a sample that includes a nucleic acid template for amplification. The nucleic acid template can be of any type, for example, genomic DNA, RNA, plasmids, bacteriophages and / or artificial sequences. The nucleic acid template can be from any source, for example, whole organisms, organs, tissues, cells, organelles (for example, chloroplasts, mitochondria), sources of synthetic nucleic acids, etc. In addition, the molds can have a wide range diversity of origins, for example, pathological samples, forensic samples, archaeological samples, etc. Biological samples may include, for example, whole blood, lymphatic fluid, serum, plasma, sweat, tears, saliva, sputum, cerebrospinal fluid (CSF), amniotic fluid, seminal fluid, vaginal excretions, serous fluid, synovial fluid, pericardial fluid , peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid, bile, urine, gastric fluids, intestinal fluids, faecal samples and swabs or washes (for example, oral, nasopharyngeal, optical, rectal, intestinal, vaginal, epidermal, etc. ) and / or other biological samples.

Various sample processing steps can be performed to prepare the nucleic acid template. In some cases, such as amplification from plasmids or bacteriophages, a crude sample will suffice. In other cases, such as amplification of large fragments from genomic DNA, highly purified template is preferred. The sample preparation steps can take place in or out of the drop microactor.

The system of the invention can be configured and programmed to allow the processing of a biological sample to prepare a drop that includes a nucleic acid template for amplification. Some or all of the parts of this processing can be carried out in or out of the drop microactor, for example, using beads having reagents attached thereto with affinity for target organisms to isolate the target organisms from a biological sample. The drop microactor can process the sample by dividing it into one or more separate drops for later operations in the drop microaction.

Samples may, in some cases, be treated to change by reducing viscosity during subsequent drop operations. For example, samples can be prepared in the drop microactor or out of the drop microactor by mixing with an alkaline solution (for example, 10% KOH) or reducing agents such as dithiothreitol (DTT) or dithioerythritol (DTE) to liquefy the sample and make it fluid enough to favor the operations of drops in a microactor of drops. Other examples of suitable sample preparation techniques are described in US Patent Application 60 / 745,950, entitled "Apparatus and Methods of Sample Preparation for a Droplet Microactuator", filed on April 28, 2006.

A drop that includes the nucleic acid template can be combined with amplification reagents to provide a drop ready for amplification, for example, combined with PCR reagents to give a drop ready for PCR. Depending on the reagents selected, the drop ready for amplification can be amplified isothermally or thermocycled to achieve amplification of a target nucleic acid. The amplified product can be detected and / or quantified in real time in a microactor of drops. In this way, the invention provides a real-time quantitative amplification on a chip for detecting and quantifying a target nucleic acid in a sample.

6.1.1.1 Primers

Reagents used in the amplification procedures of the invention will include one or more primers. In typical procedures, two primers are used to define the region of the nucleic acid template to be amplified.

The primers can also be labeled. For example, markers that facilitate the detection, location, quantification and / or isolation of the PCR product can be selected. For example, biotinylation can be used to facilitate detection and / or purification using streptavidin to capture the biotinylated PCR product on the surface. In addition, streptavidin can be associated with magnetically sensitive beads to capture the biotinylated PCR product.

6.1.2 Amplification Protocols

In one embodiment, a drop-based PCR can be run on a microaction dropper to quantify changes in gene expression levels for relevant cancer biomarkers, for example, vascular endothelial growth factor (VEGF) and dependent kinase inhibitors. of cyclin p21 (Cip1) and p27 (Kip1). For example, cells in a drop, whether suspended or bound to a surface, can be lysed. Poly (A) mRNA released in drops can be captured using beads, such as magnetically sensitive oligo (dT) beads. Reagents are available from Dynal Biotech in its DIRECT mRNA Micro Kit. Mixing or stirring of drops can be used to increase cell lysis and increase the capture of poly (A) mRNA on the beads. Magnetically sensitive oligo beads (dT) mRNA can be eluted by thermal fusion of the RNA-DNA duplex. The appropriate temperature depends on the length of the chain. The beads may be washed using drop based wash protocols as described elsewhere herein. PCR (eg, qRT-PCR) can be performed using a drop-based protocol in the drop microactor with the appropriate primers for gene targets (eg, VEGF, p21 (Cip1) and p27 (Kip1)). Drop-based RNA amplification can also be achieved using the Van Gelder and Eberwine technique.

6.2 Nucleic Acid Sequence Analysis

The invention provides methods, devices and systems for analysis of nucleic acid sequences based on drops in a microaction system of drops that, among other things, avoids the problems associated with the increasingly complex mixtures required by prior art strategies.

6.2.1 Nucleic Acid Immobilization

A sample of amplified nucleic acid can be immobilized within the microactor of drops so that the reagent drops can contact the immobilized nucleic acid. The immobilization can be on a surface of the droplet microactor or on other surfaces within the microactor, such as beads made of polymers, polymeric resins and optionally including magnetically sensitive materials.

Substrates useful for such binding include glass, gold, polyacrylamide gels, polypyrrole, Teflon and optical fibers. The materials can be provided, for example, as films, particles, matrices

or pearls In one embodiment, the substrate includes magnetically sensitive beads. The droplet microactor may include a magnet or electromagnet to produce a magnetic field to manipulate (for example, immobilize, release or move) magnetically sensitive beads. For example, magnetically sensitive beads can be agitated and immobilized on the microaction using a magnetic field to improve the washing steps (see Section 6.6).

A wide variety of techniques can be used to immobilize molecules, such as DNA, on surfaces. Examples include the chemicals used to bind oligonucleotides to the surface of microarrays and the chemicals used in solid phase synthesis techniques.

Nucleic acid samples can be thiolated and adsorbed to a gold substrate. Nucleic acids can be thiolated, for example, by substituting an internucleotide oxygen that is forming a phosphodiester moiety linkage with sulfur, for example, as described in US Patent 5,472,881, by Beebe et al., Entitled "Thiol Labeling of DNA for Attachment to Gold Surfaces", the full disclosure of which is incorporated herein by reference. One or more drops that include the thiolated nucleic acid can be transported on the droplet microactor to a gold surface, in which the thiolated nucleic acid sample will be deposited on the gold surface. The drop that includes the thiolated DNA is contacted with the gold surface for a sufficient time for covalent bonds to form between sulfur and gold. Electro-drive techniques can be used to increase the surface area of the drop with the gold surface.

A DNA sample can be biotinylated at the 5 'ends using a water soluble biotin ester or using a biotinyl phosphoramidite reagent. Biotinylated DNA can be captured on streptavidin coated substrates. Therefore, a drop that includes a sample of biotinylated DNA can be transported in contact with the surface of streptavidin in which the DNA will be captured and immobilized.

Chemistry for immobilization of single stranded DNA in a substrate has been described (S. Taira et al., "Immobilization of single-stranded DNA by self-assembled polymer on gold substrate for a DNA chip", Biotechnol Bioeng. 30 Mar 2005; 89 (7): 835-8). In this strategy, thioctic acid (TA) is covalently bonded to poly (allylamine hydrochloride) (PAH) in side chains to immobilize the polymer on a gold surface by self-assembly. Single-stranded DNA (mc) of an N-hydroxysuccinimide ester terminated probe is easily covalently immobilized on a gold surface coated with TA-PAH. The surface can be covered with polyacrylic acid, which forms ionic complexes with TA-PAH to reduce the cationic charge.

An aspect of the invention is a droplet microactor having a substrate for immobilization of a nucleic acid. Another aspect is a droplet microactor that includes a substrate for immobilization of a nucleic acid and reagents for immobilizing the nucleic acid in the substrate. Another aspect is a drop microactor that includes a substrate for the immobilization of a nucleic acid, reagents for immobilizing the nucleic acid in the substrate and a sample of nucleic acid. These reagents and samples may be stored, for example, in deposits in the drop microactor and / or in deposits or other containers outside the drop microactor (for example, in a cartridge). In one embodiment of a reservoir loading assembly, reagent and / or sample reservoirs (eg, in vials or syringes) can be coupled in fluid communication with microaction drop tanks, so that the fluid in the vials can flow or force yourself to pass directly to the microaction dropper tanks. This aspect of the invention can be scaled up, so that the number of tanks and tank load assemblies can be increased as necessary to include slots for as many reagents as necessary to perform a desired protocol.

6.2.2 Nucleotide Incorporation Facilitated by Polymerase

As illustrated in steps 4 and 5 of Figure 1, the immobilized single stranded nucleotide sample is primed to give a double stranded segment. Priming can be achieved, for example, by transporting a drop comprising a primer in contact with the immobilized sample.

The barley sample is reacted with a deoxynucleotide triphosphate (dNTP) in the presence of a polymerase. The reaction can be achieved by transporting one or more drops that include a deoxynucleotide triphosphate (dNTP) and a polymerase in contact with the immobilized sample. If the dNTP is complementary to the first base in the single stranded portion of the nucleic acid sample, the polymerase catalyzes its incorporation into the DNA chain. Each incorporation event is accompanied by pyrophosphate release (PPi) in an amount corresponding to the amount of nucleotide incorporated. Therefore, incorporation events can be determined by measuring the released PPi. The addition of dNTP is typically done one at a time, each in a separate buffer. Nucleotide incorporation takes place sequentially along each immobilized template since each nucleotide is made available in a preselected or programmed order.

A variety of native and modified polymerases can be used. Modified polymerases include, for example, native sequences with additions, insertions or substitutions that result in a polymerase that retains the ability to facilitate the incorporation of a nucleotide into a barley sample. The polymerase can be a polymerase deficient in exonuclease. The large or Klenow fragment of DNA polymerase can also be used.

A dATP or ddATP analog can be selected that does not interfere with the PPi enzymatic detection reaction but which, however, can be incorporated by polymerase into a growing DNA chain without interfering with an appropriate base pairing. Examples of suitable analogs include deoxy or dideoxy ATP [1-thio] triphosphate (or -thotriphosphate) analogs, for example, [1-thio] deoxyadenosine triphosphate or deoxyadenosine -thiophosphate (dATPS) as know too. These and other analogs are described in US Patent 6,210,891, by Nyren et al., Entitled "Method of sequencing DNA", the full disclosure of which is incorporated herein by reference for its content in relation to such analogues.

A single chain binding protein can be included that spans the length of sequences that can be sequenced by reducing the folding of the single chain sample. Therefore, for example, the invention includes a droplet microactor that includes a droplet that includes a single stranded binding protein. Gout can be a drop ready for amplification that includes a single chain binding protein. The invention includes methods of performing drop operations in a drop that includes a single chain binding protein.

6.2.3 Detection of Nucleotide Incorporation

The determination of whether a specific base has been incorporated into the target site may involve quantification of the PPi released during the incorporation reaction. As each dNTP is added to a growing nucleic acid chain during a polymerase reaction, a PPi is released. The PPi released under these conditions can be detected enzymatically, for example, by generating light in the luciferase-luciferin reaction (discussed further below). As the procedure continues, the complementary chain is assembled and the nucleotide sequence is determined from the signal peaks. The system can produce a program, as illustrated in Figure 2.

6.2.4 Drop Operation Protocols

It will be appreciated that, in addition to the protocols described above, a variety of drop operation protocols can be used to carry out the sequence analysis of the invention. Therefore, for example, conversion into ATP can be achieved in a single reaction together with the dNTP incorporation reaction, or the reactions can be carried out in steps: (1) incorporation of dNTP to release PPi, followed by (2) conversion of PPi in ATP. When the steps of incorporation and conversion are carried out together, the system can transport a single drop that includes the necessary reagents (dNTP, polymerase, ATP sulfurylase and APS). Therefore, all the reagents necessary to incorporate a dNTP into the immobilized sample, release PPi and react the PPi with APS to give ATP can be included in a reservoir in the drop microactor as a single source reagent for each nucleotide. The source reagent for each nucleotide can be successively contacted with the immobilized sample so that the reactions can take place, producing ATP in the presence of a complementary dNTP.

When the incorporation and conversion steps are performed separately, the primer, polymerase, dNTP, sulfurilase and APS can be provided from separate sources such as separate reagent drops that are joined together to perform the reactions of the invention. . Alternatively, some or all of these reagents can be provided from a single source such as a drop of pyrosequencing reagent that contacts the immobilized sample to perform the reactions of the invention. For example, a drop that includes dNTP and polymerase can be contacted with the immobilized sample, so that it will be incorporated into the base if it is complementary, thereby releasing PPi. The drop that potentially includes PPi can then be transported away from the immobilized sample and combined with a drop that includes ATP sulfurylase and APS to produce ATP. Alternatively, a drop that includes ATP sulfurylase and APS can be combined with the drop that potentially includes PPi in the presence of the immobilized sample to produce ATP.

In addition, although Figure 1 illustrates the priming stage 4 and the incorporation / conversion stages 5 as if they occur sequentially, it will be appreciated that they can be further separated.

or that can be incorporated all in one stage. In other words, each specific reagent can be added to the reaction at the appropriate time in one or more drops or any combination of multiple reagents can be provided together in a single drop or series of drops. Therefore, in one embodiment, the droplet microactor conveys one or more drops in contact with the single stranded sample, in which one or more drops together include the following reagents (or their functional equivalents) provided together or in separate drops: primer , polymerase and dNTP in any combination and in any order, producing the result that the primer hybridizes with the sample to form a double-stranded portion, the polymerase catalyzes the incorporation of any complementary dNTP at a target site in the first position of adjacent single-stranded base to the double-stranded portion, thereby releasing PPi.

In another embodiment, the drop microactor carries one or more drops in contact with the single stranded sample and the one or more drops together include the following reagents (or their functional equivalents): primer, polymerase, dNTP, sulfurylase and APS, in any combination or in any order, producing the result that the primer hybridizes with the sample to form a double stranded portion, the polymerase catalyzes the incorporation of any complementary dNTP at a target site in the first single stranded base position adjacent to the double stranded portion, freeing from this way PPi, and ATP sulfurylase convert any PPi into ATP in the presence of APS. The incorporation of bases is determined by quantification of the amount of PPi released.

The drop that potentially comprises ATP can be linked by droplet microaction techniques with a drop comprising reagents, such as luciferase and luciferin, to facilitate the detection of any ATP. Similarly, a drop that includes luciferin and potentially comprising ATP can be linked by droplet microaction techniques with a drop that includes luciferase for the detection of any ATP. In addition, a drop that includes luciferase and potentially comprising ATP can be joined by droplet microaction techniques with a drop that includes luciferin for detection of any ATP.

Drops for the detection reaction can be joined in the presence of or separated from the immobilized sample. For example, a drop of luciferase / luciferin may bind to the drop that potentially includes ATP in the presence of the immobilized sample. Alternatively, the potentially ATP drop may be separated from the immobilized sample that will bind with a drop of luciferase / luciferin. In any case, the joining of drops that include reagents that produce a light signal can be achieved in proximity to the sensor to maximize the amount of light detected.

When the drop that potentially includes ATP is transported away from the immobilized sample that will bind with a drop of luciferase, the transport stage may include an incubation step to maximize the production of ATP for detection in the fluorescence reaction. In other words, this incubation can be carried out during transport, or the drop can be temporarily stored for incubation before the fluorescence reaction. The microactor of drops may include an incubation zone for this purpose. The incubation zone may or may not include a heating element to control the temperature in the area. The incubation zone may or may not include a wall that separates the area from the rest of the microactor. The incubation zone may include an array of electrodes to facilitate the transport of drops to and from the area. The area may be scaled up and may include electrodes for transporting and storing tens, hundreds or more drops within the incubation zone.

In the practice of the invention, one or more detection reagents can be specifically excluded from the polymerase reaction step so that no signal will be emitted during the polymerase reaction. For example, in one embodiment, a detection enzyme is not added to the detection mixture for the polymerase step. Instead, the drop used to perform the polymerase stage is transported away from the immobilized sample, then attached with a drop that includes the detection enzyme in the range of a sensor to detect the signal of any resulting reaction.

The reaction mixture for the polymerase reaction may therefore include at least one dNTP, a polymerase, an APS and an ATP sulfurylase, and may optionally include luciferin, while lacking any significant amount of luciferase. In this way, the dNTP incorporation reaction can be separated from the detection reaction. The detection reaction can therefore be performed in the presence of the sensor, for example, as illustrated in Figure 2, to maximize the detection signal. For example, when the detection signal includes light, the detection reaction can be completed in the range of a sensor to detect the light emitted from any resulting light producing reaction.

In addition, a detection reaction and a subsequent incorporation reaction can be performed in parallel, thereby accelerating the sequencing rate. Similarly, an incorporation reaction can be performed, the result of a prior incorporation reaction can be incubated, and the result of a previous incubation can be subjected to detection all in parallel in separate drops, thereby accelerating the sequencing rate.

Unlike certain prior art procedures, the drop microaction strategy of the present invention avoids mass transport effects. Reagents can be contacted directly with each sample, without requiring a reagent flow through multiple samples. When magnetically sensitive beads are used, they can be held in place using a magnetic field and / or can be transported from one place to another in drops. The reagents can be transported from a reagent source directly to a sample without coming into contact with other samples and potentially causing cross contamination. The diffusion of by-products is prevented by isolation of drops in the loading fluid. Wells filled with beads, for example, stabilizing beads, which can interfere with light detection, can also be avoided. A wash containing apyrase can be used between nucleotide applications, but its use is not necessary in the practice of the invention and, in some embodiments, is specifically avoided.

Between applications of each new set of reagents to the immobilized sample, it can be washed, for example, with a buffer solution. Various surface washing protocols are described in Section 6.6.

6.3 Affinity Based Tests

The invention provides methods, devices and systems for performing affinity based drop-based assays, such as affinity-based assays. These assays include any assay in which a compound having a binding affinity for an analyte is contacted in the analyte or with a sample that potentially includes the analyte using drop operations. For example, the compound having a binding affinity for an analyte can be provided in one drop and transported in contact with an analyte that is present in another drop in a drop microactor,

or that it is immobilized on a surface of a microactor of drops. As another example, the compound that has binding affinity for the analyte can be immobilized in itself on the surface of a microactor of drops and / or on the surface of beads included in a microactor of drops, and a drop that includes the analyte or which potentially includes the analyte can contact the immobilized antibody.

It will be appreciated that a wide variety of affinity based test protocols are possible within the scope of the invention. Examples of affinity based assay formats include direct affinity based assays, indirect affinity based assays and competitive affinity based assays. The assays can be used to detect the presence of a target analyte and can also be used in some cases to quantify the target analyte present in a sample. In a competitive assay, a drop that includes a sample antibody and a labeled antibody is contacted with a surface. The antibody in the sample competes with the antibody labeled for its binding to an antigen adsorbed on the surface. A variant of this strategy involves binding the antigen to the surface by means of an intermediate crimp. For example, the linker can be an antibody. The capture bridge assay uses a drop that includes a sample antibody to bind the adsorbed antigen to the surface with solution antigen. Another strategy involves the use of biotinylated antigen and a solid phase coated with streptavidin. Another strategy involves the binding of the sample antibody to immobilized antigen on the solid phase. The bound antibody can be detected with a second labeled isotype specific antibody. Excess antibody can be washed away using the drop protocols of the invention.

It will be appreciated that an important aspect of the invention involves the ability to perform drop operations using each of the affinity reagents and / or affinity-based test samples needed in a drop microactor.

6.3.1 Samples and Sample Preparation

The invention provides drop-based affinity assays that are useful for the detection of a wide variety of analytes. For example, any analyte that can specifically bind an affinity molecule such as an antibody is suitable for detection using the systems of the invention. A single sample can be analyzed for one or more target analytes. The analytes can be, for example, biological analytes or synthetic analytes. Examples include analytes in the following categories: analytes from human sources, analytes from animal sources, analytes from plant sources, analytes from bacterial sources, analytes from viral sources and analytes from spore sources. Analytes can include, for example, proteins, peptides, small molecules and various biomolecules, such as carbohydrates, lipids and the like. One embodiment, the samples are contacted with immobilized antibody (eg, antibody immobilized on beads), prior to the introduction of the immobilized antibody on the microaction dropper.

An illustrative drop microactor 600 suitable for performing immunoassays is illustrated in Figure 3. This embodiment may employ two substrates, a first substrate 601a and a second substrate 601b separated in a substantially parallel manner to provide an intermediate space. Multiple fluid reservoirs or orifices may be provided in the intermediate space, such as wash buffer reservoirs 602, sample reservoir 604, primary antibody reservoir 606, secondary antibody reservoir 608 and immobilized antibody reservoir (eg, immobilized antibody on beads) 610. Waste areas 612 may also be provided, as well as a magnet 614 located so as to allow interaction between the magnetic field of the magnet and magnetically sensitive components located in the intermediate space. In this particular embodiment, transport electrodes 616 are provided in the first substrate.

6.3.2 Sandwich-type Affinity Based Test

In one embodiment, the invention provides a drop-based sandwich-based affinity assay performed on a microactor of drops. It will be appreciated that a wide variety of protocols are possible within the scope of the invention for performing sandwich-based affinity tests. The following drop-based protocol, which is based on the illustration in Figure 4, is provided as an example only and is not intended to limit the scope of the invention:

(one)

 immobilize on a surface an antibody (primary) with specificity by a target analyte;

(2)

 washing the immobilized antibody, for example, using a drop-based washing protocol to remove excess antibody;

(3)

 using drop operations to expose the immobilized antibody to a sample drop that potentially includes the target analyte with binding affinity for the immobilized antibody;

(4)

 washing the immobilized antibody-target analyte complex, for example, using a drop-based washing protocol to remove unbound components from the sample drop;

(5)

 exposing the immobilized antibody (which now potentially includes the target analyte bound thereto) to a drop that includes an indicator (secondary) antibody;

(6)

wash off excess indicator antibody, for example, using a drop based wash protocol;

(7)

 optionally, perform additional steps to provide a measurable parameter or signal;

(8)

 measure the measurable parameter or signal; Y

(9)

 provide an indicative result of the signal.

One or more of any of the above steps may be performed using drop operations in a microactor of drops as described herein.

6.3.2.1 Antibody Immobilization

A primary antibody is immobilized on a surface. The surface may be, for example, a surface of the droplet microactor, or the surface of beads, such as magnetically sensitive beads, non-magnetically sensitive beads, or particles, such as nanoparticles.

The immobilization of the antibody on the surface can be carried out in the microactor of drops using protocols based on drops. For example, reagents can be introduced to immobilize an antibody on a surface in the drop microactor, dispensed as separate drops and transported in contact with the surface for deposition. When the surface in question is the surface of one or more beads, the beads may be introduced into the droplet microactor, dispensed as buffer drops, transported in the droplet microactor and attached with one or more drops including reagents to immobilize the drops on the surface of the pearls.

As an alternative, the antibodies can be immobilized outside the drop microactor. For example, the antibodies can be immobilized in beads before they are introduced into the drop microactor. A variety of beads coated with protein (eg, streptavidin) and coated with antibody are commercially available. In another embodiment, the antibodies can be immobilized on a surface of the drop microactor, for example, during the manufacture of the drop microactor.

In addition, surfaces for antibody immobilization can be prepared. For example, a surface may be provided that includes moieties that have an affinity for an antibody or a binding moiety coupled to an antibody. The antibody, which optionally includes the binding moiety, can be contacted with the surface thereby immobilizing the antibody on the surface. For example, pre-coated pearls with streptavidin can be introduced into the drop microactor. Droplet operations with drops that include streptavidin-coated beads may be performed, and may be used to place such beads containing beads in contact with one or more drops that include biotinylated antibody, to thereby couple the antibody with the beads. As another example, the droplet microactor may include a streptavidin coated surface in a droplet microaction pathway, so that droplet operations can be employed to place a drop that includes biotinylated antibody in contact with the streptavidin coated surface and, of In this way, immobilize the antibody on the surface. In yet another example, capture antibodies can be selectively immobilized on the surface of the microaction dropper, for example, by designing the surface to allow the immobilization of antibodies or by coupling photoactive species directly with the antibodies or with streptavidin and, then, selectively immobilizing the antibody. by using a direct writing light system or by using an optical mask or using another means to selectively expose the light.

A wide variety of techniques are available to bind antibodies to surfaces. For example, the surface can be activated with Protein G to allow the binding of antibody molecules by their Fc domain, leaving the variable region available for target capture. The antibody can be covalently bound to latex surfaces by reaction of activated polystyrene sulfate / aldehyde activated antibody coupled with 1,3-diaminopropane. White sulfate polystyrene latex beads without surfactant can be coated with antibody by incubation with antibody and conjugation buffer (30 mM Na2CO3, 70 mM NaHCO3, pH 9.5). Biotinylated antibody can be captured on streptavidin coated substrates. The antibodies can covalently bind to a modified surface of the microaction dropper, such as a silane or thiolate layer. The antibodies can covalently bind to a modified surface of the microaction dropper (for example, during assembly or using drop operations to deposit the antibodies on the surface), such as a silane modified surface or a thiolated surface.

6.3.2.2 Binding of Diana Analyte to Immobilized Antibody

A drop of sample is contacted with the immobilized antibody to allow any target analyte present in the sample to bind with the immobilized antibody. This step can be performed using drop operations to transport a drop of sample in contact with the surface on which the antibody binds, for example, a drop that includes beads coated with antibody or a surface of the drop microactor on which it is immobilized. the antibody In an alternative embodiment, the surface is a surface of pearls and the pearl is contacted with the sample before the introduction into the microactor of drops. The pearl can also be washed before the introduction into the microactor of drops. The antibody binds to the analyte of the sample drop. The joining procedure can be accelerated by increasing the speed of mass transport. A few examples of acceleration of mass transport include the transport of the droplets at high speed to allow thorough mixing of the beads with antibodies and the target analyte, or to replenish the surface of immobilized antibody with target analytes. Other means include stirring the incubated drop in place by electric manipulation of the drop or by various external means, such as piezoelectric drive procedures.

In the absence of any means of mass transport, the binding events occur based on diffusion and could take longer times, thus prolonging the test times. The immobilized antibody (for example, the beads or the surface) may in some embodiments be subjected to a washing protocol in the microactor, for example, as described in Section 6.6, to remove excess sample or other materials.

6.3.2.3 Diana's Analyte Indicator Antibody Binding

After washing (for example, the beads or the surface), a drop comprising an indicator antibody that has an affinity for a different epitope in the analyte can be contacted with the washed immobilized antibody that potentially has the captured analyte. The labeled antibody conjugate includes an antibody coupled to an indicator molecule, such as a radioactive molecule, an enzyme capable of catalyzing a detectable reaction (e.g., a color change, chemiluminescence, fluorescence, chemiluminescence or electrochemistry), a chemiluminescent molecule or a fluorescent molecule. Depending on the indicator used, the immobilized antibody (eg, beads or other surface), which includes the analyte and indicator antibody can then be subjected to a washing protocol, for example, as described in Section 6.6, to remove excess indicator antibody.

6.3.2.4 Production and Detection of Measurable Parameters

The bound indicator antibody can be quantified by detection of a signal provided by the indicator antibody. For example, the signal may be radioactivity, color change, luminescence, fluorescence, luminescence, Raman spectrum, light scattering strategies, particle / pearl aggregation, surface plasmon resonance, Raman spectroscopic effect and the like. The detection can be direct or indirect, by detection of the amount of antibody coupled to the analyte or by detection of the amount of unbound antibody.

For strategies that require an additional reaction to produce a signal, for example, the conversion of a non-fluorescent product into a fluorescent product, a drop that includes the additional necessary reagents can be contacted with antibody-antigen-antibody complex to facilitate additional reaction

Once the indicator antibody has been allowed to bind to the analyte, excess indicator antibody can be washed away using a wash protocol, and drop operations can be used to place a drop that includes the necessary indicator reagents in contact with the immobilized antibody In one embodiment, the indicator antibody is labeled with an enzyme (for example, horseradish peroxidase (HRP) or alkaline phosphatase (ALP)) capable of catalyzing a reaction that produces a measurable parameter. For example, HRP can be used to catalyze hydrogen peroxide to generate an electrochemical signal that can be detected by measuring current or voltage. The detection of bound antibody can be achieved by a fluorescence reaction catalyzed by HRP using Amplex Red and hydrogen peroxide as substrates. Another example employs an alkaline phosphatase-mediated conversion of NBT into violet formazan, which can be detected in a colorimetrically drop. In another strategy, a chemiluminescence substrate such as luminol or Lumigen's Psatto could be catalyzed by HRP to generate a chemiluminescence signal. Other examples of suitable detection strategies are discussed elsewhere in this document.

In one embodiment, the detection step is performed in a drop in the drop microaction in the presence of a sensor to increase or maximize the signal capture from the reaction. In another embodiment, the reaction is performed away from a sensor and the drop is transported using drop operations in the presence of a sensor for detection purposes.

6.3.2.5 Alternative Sandwich Test Strategies

It will be appreciated that a variety of alternative strategies are possible. For example, the steps do not have to be performed in the order described above, for example, the indicator antibody can bind to the analyte before or at the same time the analyte is exposed to the immobilized antibody. In another strategy, the binding of capture antibody, analyte and indicator antibody can all be performed simultaneously and then presented to a capture site and then washed. In some strategies, such as Raman dispersion of enhanced surface resonance, washing is not necessary.

6.3.3 Test Based on Competitive Affinity

In one embodiment, the invention provides an assay based on competitive affinity performed on a microactor of drops. Analytes for detection by competitive affinity-based assay are typically those that are too small to bind to two antibodies, as a sandwich-type assay will be appreciated that a wide variety of protocols are possible within the scope of the invention to perform assays. based on competitive affinity. The following drop-based protocol, which is based on the illustration in Figure 5, is provided by way of illustration only and is not intended to limit the scope of the invention:

(one)

 immobilize on a surface an antibody with specificity by a target analyte;

(2)

 wash the immobilized antibody, for example, using drop-based washing protocol, to remove excess antibody;

(3)

provide a drop of sample that potentially includes target analyte and that includes an indicator analyte;

(4)

 exposing the immobilized analyte to the combined drop of target analyte / indicator analyte so that the indicator analyte competes with any target analyte for binding sites;

(5)

 wash the substrate to remove unbound analyte and indicator analyte;

(6)

 optionally, perform additional steps to produce a measurable parameter or signal; Y

(7)

 quantify the bound indicator analyte, in which the amount of indicator analyte is inversely proportional to the amount of target analyte.

One or more of any of the above steps can be performed using drop manipulation techniques in a drop microactor as described herein. Each of the stages is analyzed in more detail in subsequent sections.

6.3.3.1 Antibody Immobilization

The antibody can be immobilized as described in the section Error! The reference source is not found.

6.3.3.2 Competitive Union

A drop that includes the sample that potentially includes a target analyte is combined with a drop that includes the indicator analyte, and the combined drop is contacted with the immobilized antibody. As an alternative, the mixture of target and indicator is carried out during the loading of the microactor of drops.

The indicator analyte can be generated by coupling the indicator nucleic acid with the analyte using any of a variety of conjugation procedures. In one embodiment, the analyte portion is modified with a molecule, such as biotin, that generates a secondary capture site to immobilize a sensor DNA complex with streptavidin. Biotin coupling to the analyte should not interfere excessively with its binding to the capture antibody. The biotinylated material may in some cases also compete with the analyte of the test sample. Coupling of the biotinylated analyte with an indicator molecule can occur before or after the biotinylated analyte is captured by the immobilized antibody.

For example, in one embodiment, the assay is performed by mixing a drop with a known amount of biotinylated analyte with the sample drop potentially containing an unknown amount of unmodified analyte. A drop that includes the biotinylated analyte is combined with a drop that potentially includes the target analyte. The combined drop is contacted with the immobilized antibody, so that the biotinylated analyte and the target analyte (if any) compete for binding to the immobilized antibody. The amount of biotinylated analyte bound is inversely proportional to the amount of analyte in the test drop. The immobilized antibody (for example, the beads or the surface) can then be subjected to a washing protocol, for example, as described in Section 6.6, to remove excess of the indicator analyte.

6.3.3.3 Indicator Analyte Detection

After washing, a drop with an indicator complex with streptavidin-biotin is added a drop that includes the washed or otherwise contacted beads with a surface that includes the immobilized antibody. The streptavidin-biotin indicator complex binds to any biotinylated analyte that had been captured by the antibody in the bead.

6.4 Immuno-PCR

The invention provides a drop-based immuno-PCR (Pick) for sensitively detecting analytes that are available only at trace levels. The present invention combines the various affinity-based assay media that use a detector antibody on a drop-based platform and utilizes a nucleic acid chain as a marker. This nucleic acid chain is amplified using amplification techniques (see, for example, Section 6.1).

It will be appreciated that an important aspect of the invention implies the ability to perform drop operations using each of the necessary iPCR reagents and / or samples in a drop microactor. For example, the invention includes:

(one)

 a droplet microactor comprising therein a droplet comprising one or more of any of the reagents and / or samples described herein for performing iPCR protocols;

(2)

 a device or system of the invention comprising said droplet microactor;

(3)

 a method of carrying out operations of drops in, or otherwise manipulating a drop, which makes use of said system or microactor of drops; I

(4)

 a method of conducting a drop-based affinity-based assay that makes use of said droplet system or microactor.

6.4.1 iPCR sandwich type

In one embodiment, the invention provides a drop-based sandwich iPCR made in a microactor of drops. In general, sandwich-type iPCR implies:

(one)

 immobilize on a surface an antibody with specificity by a target analyte;

(2)

 washing the immobilized antibody, for example, using a drop based wash protocol;

(3)

 exposing the immobilized antibody to a drop of sample that potentially includes the target analyte;

(4)

 washing the immobilized antibody, for example, using a drop based wash protocol;

(5)

 exposing the immobilized antibody (which now potentially includes the target analyte bound thereto) to a drop that includes a second antibody conjugated to a nucleic acid molecule:

(6)

 washing the immobilized antibody, for example, using a drop-based test protocol;

(7)

 amplify the nucleic acid and detect the amplification (if any) to determine the presence and amount of target analyte captured.

One or more of any of the above steps can be performed using drop manipulation techniques in a drop microactor as described herein. Each of the stages is analyzed in more detail in subsequent sections.

6.4.1.1 Antibody Immobilization

A primary antibody is immobilized on a surface. The surface may be, for example, a surface of the droplet microactor, or the surface of beads, such as magnetically sensitive beads. The immobilization of the antibody on the surface can be carried out in the microactor of drops using protocols based on drops. For example, reagents for immobilizing an antibody on a surface can be introduced into the drop microactor, dispensed as separate drops and transported in contact with the surface for deposition. When the surface in question is the surface of one or more beads, the beads may be introduced into the drop microactor, dispensed as drops in a buffer, transported and bound with one or more drops including reagents to immobilize the drops on the surface of the pearls.

A wide variety of techniques are available for antibody binding to surfaces. For example, the surface can be activated with Protein G to allow the binding of antibody molecules through its Fc domain, leaving the variable region available for target capture. The antibody can be covalently bound to latex superpies by reaction of activated polystyrene sulfate / aldehyde activated antibody coupled with 1,3-diaminopropane. White sulfate polystyrene latex beads without surfactant can be coated with antibody by incubation with antibody and conjugation buffer (30 mM Na2CO3, 70 mM NaHCO3, pH 9.5). Biotinylated antibody can be captured on streptavidin coated substrates. A light-directed immobilization can also be performed, for example, as described elsewhere in the present disclosure.

6.4.1.2 Binding of Diana Analyte to Immobilized Antibody

A drop of sample is contacted with the immobilized antibody to allow any target analyte present in the sample to bind with the immobilized antibody. This step can be carried out by transporting a drop of sample in contact with a surface of the drop microactor on which the antibody is immobilized. In another embodiment, the step can be performed by joining a drop of sample with a drop that includes beads on which the antibody has been immobilized. The antibody binds to the analyte of the sample drop. The immobilized antibody (eg, beads or surface) can be subjected to a washing protocol, for example, as described in Section 6.6.

6.4.1.3 Antibody-NA binding to Diana Analyte

After washing (for example, the beads or the surface), a drop comprising an antibody-NA conjugate that has an affinity for a different epitope in the analyte can be contacted with the washed immobilized antibody that potentially has the captured analyte . The antibody-NA conjugate includes a nucleic acid molecule coupled to the antibody. The nucleic acid molecule serves as a nucleic acid template for amplification. The immobilized antibody (eg, beads or surface) is then subjected to a wash protocol, for example, as described in Section 6.6.

6.4.1.4 Nucleic Acid Amplification

The nucleic acid is amplified, for example, as described in Section 6.1. The amount of amplified product produced is measured, for example, using real-time fluorescence detection, electrochemical and / or electrochemiluminescent detection. The amount of PCR product produced is correlated with the amount of antibody-bound DNA, which in turn depends on the amount of analyte present in the sample drop.

6.4.1.5 Alternative Strategy

In an alternative embodiment, the order of these steps is generally reversed to perform nucleic acid amplification followed by an affinity-based assay that results in an optical or electrical signal. In this sequence, an immunoassay would be performed to control the amplification of nucleic acid.

6.4.2 Competitive iPCR

In one embodiment, the invention provides a competitive iPCR performed on a microactor of drops. Analytes for competitive iPCR detection are typically those that are too small to bind to two antibodies, as required by a sandwich type assay. In general, competitive iPCR implies:

(one)

 immobilize on a surface an antibody with specificity by a target analyte;

(2)

combining a drop of sample that potentially includes target analyte with a drop that includes an indicator analyte;

(3)

exposing the immobilized antibody to the combined drop of target analyte / indicator analyte, so that the indicator analyte competes with any target analyte for binding sites;

(4)

 wash the substrate to remove unbound analyte;

(5)

 coupling the indicator analyte bound to an indicator nucleic acid; Y

(6)

 amplify the indicator nucleic acid and control the development of the amplification to determine the amount of unbound indicator analyte, which for the bound indicator analyte is inversely proportional to the amount of target analyte in the sample.

One or more of any of the above steps can be performed using drop manipulation techniques in a drop microactor as described herein. Each of the stages is analyzed in more detail in subsequent sections.

6.4.2.1 Competitive Union

A drop that includes the sample that potentially includes a target analyte is combined with a drop that includes the indicator analyte and the combined drop is contacted with the immobilized antibody.

The indicator analyte can be generated by coupling the indicator nucleic acid with the analyte using any of a variety of conjugation procedures. In one embodiment, the analyte portion is modified with a molecule, such as biotin, that generates a secondary capture site to immobilize a streptavidin-DNA complex. The coupling of biotin with the analyte should not interfere excessively with its binding to the primary capture antibody. The biotinylated material may in some cases also compete with the analyte in the sample. Coupling of the biotinylated analyte with the indicator nucleic acid can occur before or after the biotinylated analyte is captured by the immobilized antibody.

For example, in one embodiment, the assay is performed by mixing a drop with a known amount of biotinylated analyte with the sample drop potentially containing an unknown amount of unmodified analyte. A drop that includes the biotinylated analyte is combined with a drop that potentially includes the target analyte. The combined drop is contacted with the immobilized antibody, so that the biotinylated analyte and the target analyte (if any) compete for binding to the immobilized antibody. The amount of biotinylated analyte bound is inversely proportional to the amount of analyte in the sample drop.

6.4.2.2 Coupling to the Nucleic Acid Indicator

After washing, a drop with a streptavidin-biotin indicator nucleic acid complex is added to a drop that includes the washed beads or surface. The streptavidin-biotin indicator nucleic acid complex binds to any biotinylated analyte that had been captured by the antibody in the bead.

6.4.2.3 Nucleic Acid Amplification

After washing, the amount of an indicator nucleic acid complex with immobilized streptavidin biotin is determined by amplification of the indicator nucleic acid. The amplification signal is an inverse measure of the amount of analyte in the original sample. Amplification can be developed as described in Section 6.1. The presence and quantity of amplified product produced are measured, for example, using real-time fluorescence detection. The amount of indicator analyte that was displaced is proportional to the amount of target analyte in the sample.

6.4.2.4 Alternative Strategies

The stages are not limited to the given order. For example, the antibody can bind to the target analyte / indicator analyte before it is immobilized by combining a drop that includes the free antibody with one or more drops that include the target analyte / indicator analyte, after which the antibody can be placed. in contact with the surface for immobilization. The indicator analyte can be conjugated with the indicator nucleic acid in the drop microactor by combination of drops that include the two reagents. A drop that includes the indicator analyte can be combined with a drop that includes the indicator nucleic acid to effect conjugation before or after the indicator analyte is exposed to the captured antibody. A variety of alternatives are possible within the scope of the invention.

6.4.3 Samples and Sample Preparation

A wide variety of analytes can be detected using drop-based iPCR protocols of the invention. A single sample can be analyzed for one or more target analytes. The analytes can be, for example, biological analytes or synthetic analytes. For example, in one embodiment, the analytes are selected from the following categories: bacterial source analytes, viral source analytes, spore source analytes, protein toxin analytes and low molecular weight toxin analytes. In one embodiment, the target analytes include toxins or analytes indicative of the presence of specific biological organisms, for example, infectious diseases, or toxins that are employed in bioterrorism or biological warfare. Examples of such organisms include anthrax, avian influenza, botulism, hantavirus, legionellosis, pneumonic plague, smallpox, tularemia and viral hemorrhagic fevers (FHV).

6.4.4 Immuno-PCR protocols

The system of the invention allows multiple immuno-PCR assays to be performed simultaneously in a single sample. In addition, immuno-PCR assays can be performed in conjunction with other assays, such as PCR and / or affinity based assays.

6.5 Multi-Analyte Analyzer

Various protocols for nucleic acid amplification, nucleic acid sequencing, affinity-based assays, cell manipulation, pearl manipulation and washing, and analyte detection protocols can also be easily integrated into a single microaction drop system. In one embodiment, a single droplet microactor includes reagents and detection components to perform nucleic acid amplification and nucleic acid sequencing. In another embodiment, a single droplet microactor includes reagents and detection components to perform nucleic acid amplification for the detection of a blood pathogen and reagents and detection components to perform one or more tests other than test types and / or for analyte types as described herein. In another embodiment, a droplet microactor includes components for handling cells together with components and reagents for affinity-based assays.

6.6 Surfaces and Surface Washing Protocols

Various protocols of the invention require surfaces for immobilization of reagents. For example, surfactants can be used to capture or immobilize target components of a drop, such as cells, other beads, microparticles, nanoparticles, antibodies, proteins, peptides, nucleic acids, small molecules and / or other chemical components. Surfaces used for such purposes may include, for example, surfaces of beads, microparticles, nanoparticles, membranes, physical objects and / or micro-actuator surfaces of droplets. Various protocols require a washing step in which unbound materials are removed from one or more surfaces.

A sample drop that includes one or more target components for capture can be contacted, using drop operations, with a surface that has affinity for said targets. Washing protocols of the invention can be used to remove unbound components from the sample drop from the surface. For example, a drop protocol can be used to place one or more drops that include one or more target components in contact with one or more surfaces, so that one or more target components can be immobilized or captured on the one or more surfaces. A washing protocol can be executed to remove unbound substances from the one or more surfaces. Similarly, a drop protocol can be used to place one or more drops that include one or more target components in contact with one or more beads, so that the one or more target components can be immobilized or captured on the one or more beads . A washing protocol can be executed to separate unbound substances from the one or more beads.

Washing generally involves placing one or more washing drops in contact with the immobilized surface. Washing may involve agitation of the drops while in contact with the surface. Washing drops may include, for example, water, deionized water, saline solution, acid solutions, basic solutions, detergent solutions and / or buffers.

The washing protocols of the invention result in a highly effective removal of unbound substances from the surface. In one embodiment, the invention provides a method of providing a drop in contact with a surface with a reduced concentration of a substance. This process may generally include providing a surface in contact with a drop comprising a starting concentration of the substance, and having a starting volume; perform one or more operations of drops to join a drop of washing with the drop to give a combined drop; and performing one or more drops operations to divide the combined drop to give a set of drops that includes: (i) a drop in contact with the surface having a decreased concentration and a decreased amount of the substance relative to the starting concentration ; and (ii) a drop that is separated from the surface.

The process of the invention may give a drop in contact with the surface having a decreased amount or substantially decreased amount of the substance with respect to the starting concentration. The resulting drop may in some embodiments have a volume that is approximately the same as the starting volume. In some embodiments, the washing steps may be repeated until a predetermined maximum amount of the one or more components in the resulting drop is reached or exceeded. The predetermined amount may represent a substantial reduction with respect to the starting concentration. In some cases, the resulting drop may be substantially free of the components. For example, in some embodiments, the amount reduction exceeds 99, 99.9, 99.99, 99.999, 99.9999, 99.99999, 99.999999 percent on a molar basis.

The process of the invention can produce a drop in contact with the surface having a decreased concentration or a substantially decreased concentration of the substance with respect to the starting concentration. The resulting drop may in some embodiments have a volume that is approximately the same as the starting volume. In some embodiments, the washing steps may be repeated until a predetermined maximum concentration of one or more components in the resulting drop is reached or exceeded. The predetermined concentration limit may represent a substantial reduction with respect to the starting concentration. In some cases, the resulting drop may be substantially free of the components. For example, in some embodiments, the reduction in concentration exceeds 99, 99.9, 99.99, 99.999, 99.9999, 99.99999, 99.999999 percent.

6.6.1 Pearl Wash

For protocols that make use of beads, a drop with beads can be combined using drop operations with one or more wash drops. Then, while retaining the beads (for example, physically or magnetically), the attached drop can be divided using drop operations into two or more drops: one or more drops with beads and one or more drops without a substantial amount of beads. In one embodiment, the attached drop is divided using drop operations in a drop with beads and a drop without a substantial amount of beads.

Generally, each execution of a wash protocol results in the retention of sufficient beads to perform the desired test without excessively detrimental effects on the test results. In certain embodiments, each division of the bound drop results in the retention of more than 90, 95, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99, 6, 99.7, 99.8, 99, 99.9, 99.99, 99.999, 99.9999, 99.99999 or 99.999999 percent pearls. In other embodiments, each execution of a wash protocol to achieve a predetermined reduction in the concentration and / or amount of the substance removed results in the retention of more than 99, 99.1, 99.2, 99.3, 99 , 4, 99.5, 99.6, 99.7, 99.8, 99, 99.9, 99.99, 99.999, 99.9999, 99.99999 or 99.999999 percent pearls. In yet other embodiments, the amount of retained beads is calculated and the results are adjusted accordingly.

In some embodiments, the beads may be washed in tanks in which the drop containing beads and the wash drops are combined, the beads are retained (for example, by a magnet, by physical structures, electrostatic forces), and the drops that No pearls are dispensed from the tank using drop operations. For example, the beads can be washed by a dilution and dispensing strategy whereby a wash buffer is added to the tank to dilute the content, the magnetically sensitive beads are located inside the tank with a magnet and most of the solution is dispensed from the tank, and this cycle is repeated until acceptable levels of washing are reached.

A non-limiting example illustrated in Figure 6 involves immobilizing magnetically sensitive beads using a magnetic field. Magnetically sensitive immobilized beads can be released by reduction or elimination of the magnetic field. Washing of magnetically sensitive beads can generally include the following steps:

(one)

 using drop operations to place a drop 1101 comprising magnetically sensitive beads 1102 and unbound substances 1103 in proximity to a magnet 1104;

(2)

 using drop operations to combine a wash drop 1106 with the drop 1101 comprising the magnetically sensitive beads 1102;

(3)

 immobilize the beads 1102 by application of a magnetic field;

(4)

 using drop operations to remove some or all of the drops surrounding the beads to give a drop 1108 comprising the beads with a reduced concentration of unbound target substance and a drop 1110 comprising a unbound target substance;

(5)

 release the beads 1102 by removing the magnetic field;

(6)

 Repeat steps (2) to (3) or (2) to (4) until a predetermined degree of purification is achieved.

In this way, unbound substances, such as contaminants, byproducts or excess reagents, can be separated from the beads. Each cycle produces a drop that includes pearls but with a decreased level of unwanted substances. Step (5) is not necessary in each wash cycle; however, it may be useful for improving the release wash of contaminants that may be trapped in immobilized beads. The stages can be performed in a different order, for example, the steps (2) and (3) can be reversed. The steps in the washing protocol can be carried out in a microactor of drops using operations of drops as described herein.

Another embodiment is illustrated in Figure 7 and may comprise an upper plate 1201, a lower plate 1202, electrodes 1203 and a magnet 1204. The steps of the embodiment may generally include:

(one)

 using drop operations to combine an ingot 1205 with a drop 1206 comprising magnetically sensitive beads 1207 and unbound material 1208 in proximity to a magnet 1204;

(2,3) with the 1207 beads immobilized, use drop operations to transport the resulting combined ingot 1210 through the beads 1207 to separate the unbound material 1208 from the beads 1207;

(4)

 using drop operations to remove a portion of the combined ingot 1210 separately to give a portion 1212 comprising the beads with a reduced concentration of unbound target substance and a portion 1214 comprising unbound target substance;

(5)

 Repeat steps (1) - (4) as necessary to achieve the desired reduction in unbound material.

In a related strategy, the ingot can be continuously supplemented by the addition of additional wash drops and / or ingots as the ingot is transported through the immobilized beads. The procedure can continue until the desired reduction in unbound material is achieved.

Figure 8 illustrates an alternative embodiment that may also comprise an upper plate 1301, a lower plate 1302, electrodes 1303 and a magnet 1304. In this embodiment, the magnet 1304 moves, such as in the direction of A1, to separate the beads. 1305 of unbound material 1306 in a combined ingot 1307 instead of moving the ingot 1307. A similar strategy involves the movement of both the magnet and the ingot to achieve separation (not shown). Another strategy involves the use of multiple magnets to move the pearls (not shown).

In embodiments in which magnetically sensitive beads are used, the inventors have discovered that the application of a magnetic field, although useful for temporarily immobilizing the beads, moving the beads and / or locating the beads, sometimes results in an aggregation not Desired pearls. In one embodiment, a surfactant is included to prevent or reduce pearl aggregation. Examples of suitable surfactants for this purpose include: Tween®20, Tween®80, Triton X-100. Surfactants should be selected and used in amounts that reduce or eliminate pearl aggregation and minimize nonspecific adsorption while at the same time not resulting in a significant loss of target analytes or drop reagents.

Another strategy to eliminate or reduce aggregation by pearl agglutination involves the use of small amounts of larger pearls. Any amount of beads that may be contained in a drop can be used during one or more drop operations. In some embodiments, the number of magnetically sensitive beads may vary from one to several hundred thousand. For example, in one embodiment, the invention makes use of one to 100 magnetically sensitive beads per drop. For example, the invention can make use of 1, 2, 3, 4, 5, 6. 7, 8, 9, 10, ... 100 magnetically sensitive beads per drop. In one embodiment, the number of magnetically sensitive beads is from one to 10. The use of smaller amounts of magnetically sensitive beads allows larger beads to be used. For example, in one embodiment, the invention makes use of one to 100 magnetically sensitive beads per drop, in which the beads have an average diameter of about 25 to about 100 micrometers. In another embodiment, the invention makes use of one to 10 magnetically sensitive beads per drop, in which the beads have an average diameter of about 50 to about 100 micrometers.

6.7 Cell Handling

Immunogenic capture of relevant cells can be performed using antibody beads, such as anti-cytokeratin beads, and can be used to capture relevant cells from a sample before being introduced into the drop microactor and / or from a drop in a drop. drop microaction. Binding can be increased or incubation times can be reduced in the drop microactor by actively moving the drop or vortexing the drop into a reservoir. The beads can be isolated and washed as described elsewhere herein. The target cells can be released in a suspension in a drop in the drop microactor.

6.8 Architecture and Operation of the Drop Microactor

The system of the invention generally includes a droplet microactor controlled by a processor. For example, the processor can, among other things, be programmed to control drop manipulations in a drop microactor. A wide variety of configurations of droplet micro-actuators are possible. An illustration is provided in Figure 3. Examples of components that can be configured in a droplet microactor of the invention include various loading fluids that can be loaded into the droplet microactor; fluid loading mechanisms for introducing loading fluid, sample and / reagents into the microactor actuator; various deposits, such as entry deposits and processing deposits; drop dispensing mechanisms; means for controlling the temperature of the droplet microactor, of the loading fluid and / or of a drop in a droplet microactor; and magnetic field generating components for manipulating magnetically sensitive beads in a microactor of drops. This section discusses these and other aspects of the drop microactor and its use in the systems of the invention.

6.8.1 Drop Microactor

The systems make use of a microactor of drops. The drop microactor will include a substrate with one or more electrodes arranged to perform one or more drop operations. In some embodiments, the drop microactor will include one or more series, pathways or networks of said electrodes. A variety of electrical properties can be used to perform drop operations. Examples include electrohumidification and electrophoresis.

Various components of the invention can be included as components of the droplet microactor. In fact, a whole system of the invention can be provided as an integrated drop microactor. In some embodiments, the drop microactor includes various sensors and means for electronically coupling the sensors to external circuits. In other embodiments, the microactor of drops includes heaters and / or magnetic field generating elements and means for coupling said elements to external circuits. In addition, a drop microactor that includes one or more of any of the reagents described herein in a reservoir or in a drop form is also an aspect of the invention.

6.8.1.1 Drop Operations

The microactor of drops can perform various operations of drops with respect to a drop. Examples include: loading a drop into the drop microactor; dispense one or more drops from a drop of source; split, separate or divide a drop into two or more drops; transport a drop from one location to another in any direction; join or combine two or more drops in a single drop; dilute a drop; mix a drop, shake a drop; deform a drop; hold a drop in position; incubate a drop; heat a drop; vaporize a drop; cool a drop; get rid of a drop; transport a drop out of a drop microactor; other drop operations described herein; and / or any combination of the above.

Excision of drops or division of drops generally involves separating a drop into two or more sub-drops. In some cases, the resulting drops are relatively the same size.

Transportation involves moving a drop from one location to another in any direction. The drops can be transported in a plane or in three dimensions. It will be appreciated that a variety of droplet operations, such as dispensing and / or excision, may include a transport element, in which one drop is transported away from another drop. The union involves combining two or more drops in a single drop. In some cases, drops of relatively the same size are joined together. In other cases, a drop can be joined in a larger drop, for example, by combining a drop with a larger volume present in a reservoir.

The invention includes drops operations using beads comprising beads. A variety of such operations are described elsewhere in this document. In one embodiment, beads are used to perform drop operations on reagents that tend to interfere with the drop operations. For example, certain proteins may tend to bind to surfaces of a droplet microactor and / or to be distributed in the loading fluid. The immobilization of said compounds in hydrophilic beads can be used to facilitate the operations of drops using the compounds. The compounds may be attached to the beads and the beads may be contained in a drop that is subjected to drop operations.

6.9 Systems

The fluid loading can be performed using micro-actuator drop systems as illustrated in Figure 9. The steps of a fluid loading protocol can be performed using a drop control system 1801. A set of computer-executable instructions can be written that they can be loaded into a controller for the execution of a loading protocol. Integrated systems that include the drop control system 1801 and the protocol execution system 1802 can also be used. The drop control system 1801 allows a user to control the functions of the microaction drop system, such as drop operations and Sensor operations for fluid loading protocols. The protocol execution system 1802 allows a user to execute software routines that control the functions of the microaction drop system, such as drop operations and fluid loading operations. The invention also provides a method or instructions usable by computer for performing protocols or procedures for fluid loading. The programmable flexibility of the platform allows trials to be optimized quickly and allows conditional execution stages to be implemented. For example, calibrations, confirmation tests or additional controls can be executed if triggered by a particular test result. In some embodiments, the system may integrate sample preparation steps. The automation of the system and the operations of the microactor in drops improves portability and allows the tests to be carried out more quickly and by personnel with minimal training, thus reducing human errors.

Referring further to Figure 9, at a high level, each of the systems of the invention typically includes a processor or controller 1803, a drop microactor 1804, a sensor or detector 1805, an input device or devices 1806, a 1807 output device or devices and software. US Patent Application No. 60 / 806,412, entitled "Systems and Methods for Droplet Microactuator Operations", filed on June 30, 2006, the full disclosure of which is incorporated herein by reference, describes micro-actuator droplet systems that they can be used in conjunction with the droplet microactor aspects of the invention. The drop control system includes a drop control software processed on a computer 1808 and programmed to deploy a drop control interface to control the functions of the microaction drop system. The protocol execution system includes a programmed protocol execution software to facilitate the execution of a set of instructions executable by computer or usable by computer to control the functions of the microactioner drop system to carry out fluid loading.

6.9.1 Controller

The system of the invention may include a controller 1803. The controller serves to provide processing capabilities, such as storing, interpreting and / or executing software instructions. The controller can be, for example, composed of a digital signal processor (DSP) with memory, a microcontroller or a specific application integrated circuit (ASIC). An example of a suitable DSP processor is the DSP Analog Devices Blackfin processor.

The controller is electronically coupled to various hardware components of the invention, such as the drop microactor, any sensor and any input and / or output device. The controller can be configured and programmed to control data and / or energy aspects of these devices. For example, with respect to the microactor of drops, the controller controls the manipulation of drops by activation / deactivation of electrodes. This aspect of the invention is further analyzed in Section 6.8.

The controller may also be electronically coupled to a separate computer system that includes a processor, input and output devices, a data storage medium and other components. This arrangement is particularly useful in the drop control system, in which the computer system is programmed to operate a drop control user interface. In this arrangement, the processor of the computer system can support data entry through the user interface and transmit the instructions to the controller, for example, to activate / deactivate the electrodes, to read the electrodes, memory and / or the sensors and the like

In the protocol execution system, software to control the system can be loaded directly into and executed by the controller to cause the controller to control the functions of the microactioner drop system. In this embodiment, the system can operate autonomously, for example, as a handheld or portable system.

6.9.2 Drop Microactor

The system may include a 1804 microaction actuator, as further described in Section 6.8. The drop microactor is electronically coupled to the processor, so that the processor can control various operations of the drop microactor, such as drop manipulation operations.

6.9.3 Sensor

Various embodiments of the invention make use of sensors or detectors 1805. The sensors may include sensors that are coupled to the droplet microactor in order to measure parameters of interest in the droplet microactor, such as fluorescent or luminescent intensity at a location in the microactor of drops in which a reaction product can be located. The sensors can also include sensors that control the state of the system, such as microactivator insertion sensors, cover clamp sensors, ambient temperature sensors and the like. The output of each sensor can be mapped to a specific memory location and the processor only has to check the mapped location to obtain a sensor reading. The sensor is mounted with respect to the drop microactor and / or electronically coupled to the drop microactor, so that the sensor can detect signals, such as light or electrical signals, from the drop microaction.

6.9.4 Input or Output Device or Devices

The systems of the invention also include various input devices 1806 and output devices 1807. In certain embodiments, such as the protocol execution system, certain input and output devices can be controlled using a man-machine interface (HMI) controller. .

6.9.5 Software

Each of the systems of the invention includes software. The software provided in a storage medium is an aspect of the invention. Examples of suitable storage media include magnetic storage, optical storage, phase change memories, holographic storage, molecular memory storage, battery or capacitor backed SRAM and flash memory storage. The software can be loaded into a memory and / or a processor. An aspect of the invention is also a system in which the software of the invention is present in a memory and / or a processor and / or a storage medium.

The software of the invention can be written in any of a variety of programming languages, such as Visual C, Java and / or Python. The system can include an interpreter to translate the manipulation of drops and other instructions from the high level language to an intermediate language for execution by the processor. Alternatively, software written according to the invention can be compiled in machine language using a compiler. The software interpreter and the compiler for the language of the invention are themselves novel aspects of the invention. As such, all forms of data storage, memories and processors containing the interpreter and / or compiler are aspects of the invention.

The system can be programmed to execute a wide diversity of protocols that involve any number of drop manipulations. Multiple drops can be handled independently and simultaneously in a single drop microactor. The ability to independently manipulate multiple drops in parallel allows the execution of complex protocols such as a series of basic microfluidic instructions. The systems can be scaled up and can control tens, hundreds, thousands or more drop manipulations in parallel by drop microaction. For example, at any time, up to a maximum of each control electrode in the microaction actuator can be engaged in a droplet operation.

The system can be programmed to allow users to enter instructions for protocol execution. Existing protocols can be controlled and adjusted according to the user's needs. Complex protocols can be implemented in which the result of a

or more stages determines the selection of one or more subsequent stages. For example, a drop in which a certain measured result is positive can be transported for further processing, while a drop in which a result is negative can be discarded or vice versa.

6.9.6 Portability

With respect to Figures 10A and 10B, in some embodiments, the analyzer is provided as a portable device, such as a handheld device 1900. Figure 10A shows the exterior of handheld device 1900 and Figure 10B shows a slot 1902 for the insertion of a drop microactor (not shown), an optical sensor 1904 to detect optical signals from the drop microactor and a cover fastener 1906, which can be coupled to the system to indicate whether the lid is open or closed. It is envisioned that the portable analyzer can also be a desktop device. The portability of the micro-actuator drop systems of the invention facilitates the use at the point of assistance or at the point of sample collection in a wide variety of clinical settings, operating rooms, emergency rooms, small laboratories and in the field (medical equipment emergency response, accidents, disasters, battlefields, bioterrorism sites, etc.) for a quick diagnosis, which can lead to turning times quickly in critical situations.

6.10 User Interface

The drop control system includes a drop control software programmed to deploy a drop control interface to control the drop operations in the drop microactor, control the sensor, when present, and control other hardware associated with the drop system. drop control The system may also include software to facilitate the creation of a set of software or instructions usable by computer to control the functions of the microactioner drop system, such as drop operations and / or sensor operations.

6.10.1 Protocol Execution System and User Interface

The invention provides a protocol execution system. The protocol execution system includes a programmed protocol execution software to facilitate the execution of a set of software instructions for loading fluid, controlling drop operations in the drop microactor and / or other functions of a drop microactor and the related hardware. The protocol execution system provides the ability to execute protocols in a separate system, typically a handheld or portable system.

The protocol execution system is configured to control the drop microactor and any associated components. Preprogrammed instructions can be loaded into the controller that controls the system and any associated components. The protocol execution system may include various components to allow a user to provide data entry and obtain a processor result. The man-machine interface can be facilitated using an HMI card. The HMI card typically includes a controller and various electronic components, such as buses and ports to electronically couple input and output devices with the processor.

6.11 Concluding Observations

The above detailed description of embodiments refers to the accompanying drawings, which illustrate specific embodiments of the invention. Other embodiments having different structures and operations do not depart from the scope of the present invention.

As one skilled in the art will appreciate, the present invention can be incorporated as a software program, system or product. Accordingly, the present invention may take the form of a fully hardware embodiment, a completely software embodiment (including firmware, resident software, microcode, etc.) or an embodiment that combines aspects of software and hardware that can all be termed in general in this document "circuit", "module" or "system". In addition, the present invention may take the form of a computer program product in a computer-usable storage medium that has a computer-usable program code incorporated in the medium.

Any suitable computer usable media can be used. The computer-usable or computer-readable medium may be, for example, but not limited to a system, apparatus, device or electronic, magnetic, optical, electromagnetic, infrared or semiconductor propagation medium. More specific examples (a non-exhaustive list) of the computer-readable medium would include some or all of the following: an electrical connection that has one or more cables, a laptop diskette, a hard disk, a random access memory (RAM) , a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), an optical fiber, a portable compact disc read-only memory (CD-ROM), an optical storage device, a transmission medium such as those that support the Internet or an intranet, a magnetic storage device. Note that the computer-usable or computer-readable medium may even be paper or other suitable media on which the program is printed, since the program can be captured electronically, for example, by optical scanning of the paper or other media, then compiled, be interpreted or otherwise processed in an appropriate manner, if necessary, and then stored in the memory of a computer. In the context of this document, a computer-usable or computer-readable medium may be any medium that may contain, store, communicate, propagate or transport the program for use by or in connection with the system, apparatus or device for executing instructions.

The code of the computer program for carrying out the operations of the present invention may be written in an object-oriented programming language such as Java, Smalltalk, C ++ or the like. However, the code of the computer program for carrying out the operations of the present invention may also be written in conventional procedural programming languages, such as the "C" programming language or similar programming languages. The program code can be executed entirely on the user's computer, partially on the user's computer, as a stand-alone computer package, partially on the user's computer and partially on a remote computer, or entirely on the computer or server remote. In the latter case, the remote computer can be connected to the user's computer through a local area network (LAN) or a wide area network (WAN), or the connection can be made with an external computer (for example, through Internet using an Internet Service Provider).

The present invention is described with reference to illustrations of flowcharts and / or block diagrams of procedures, apparatus (systems) and software products in accordance with embodiments of the invention. It will be understood that each block of the flowchart illustrations and / or block diagrams, and block combinations in the flowchart illustrations and / or block diagrams, may be implemented by computer program instructions. These computer program instructions may be provided to a processor of a general purpose computer, special purpose computer or other programmable data processing apparatus to produce a machine, so that the instructions, which are executed through the computer processor or another programmable data processing apparatus, create means to implement the functions / acts specified in the block or blocks of the flow chart and / or block diagram.

These computer program instructions can also be stored in a computer-readable memory that can be directed to a computer or other programmable data processing apparatus to operate in a particular way, so that the instructions stored in the computer-readable memory produce a article of manufacture that includes instructional means

5 that implement the function / act specified in the block or blocks of the flowchart and / or block diagram.

The computer program instructions can also be loaded on a computer or another

programmable data processing apparatus to cause a series of steps to be performed

functional in the computer or other programmable device to produce an applied procedure

10 informatically, so that the instructions executed in the computer or other programmable apparatus provide steps to apply the functions / acts specified in the block or blocks of the flow chart and / or block diagram.

The present specification is divided into sections for the convenience of the reader only. Headings should not be construed as limiting the scope of the invention.

It will be understood that various details of the present invention can be changed without departing from the scope of the present invention. In addition, the above description is for illustrative purposes only and not for the purpose of limiting, since the present invention is defined by the claims set forth hereinbelow.

Claims (6)

1. A method for providing a drop in contact with a surface of one or more magnetically sensitive beads (1102, 1207, 1305) comprised in the drop in a microactor of drops, with a reduced concentration of the substance, the process comprising:

(to)

providing a drop microactor comprising said surface of one or more magnetically sensitive beads in contact with the drop (1101, 1206), the drop comprising a starting concentration and a starting amount of a substance (1103, 1208, 1306) and having a starting volume;

(b)

 performing one or more droplet operations to join a wash drop (1106, 1205) with the drop (1101, 1206) provided in step (a) to give a combined drop (1307); Y

(C)

 immobilize the one or more magnetically sensitive beads by exposing said one or more magnetically sensitive beads to a magnetic field;

characterized in that while said one or more magnetically sensitive beads are thus immobilized, said method further includes the step of:

(d)

 Perform one or more operations of electrohumectation-mediated drops to divide the combined drop to give a set of drops comprising:

(i)

 a drop (1108, 1212) in contact with said surface of one or more magnetically sensitive beads having a decreased concentration of the substance with respect to the starting concentration; Y

(ii)

 a drop (1110, 1214) that is separated from said surface of one or more magnetically sensitive beads.

2.

The method of claim 1, further comprising releasing or resuspending one or more of the beads after step (d) of claim 1.

3.

The method of claim 1, wherein step (c) comprises placing the drop (1101, 1206) comprising the magnetically sensitive beads in proximity to a medium (1104, 1204, 1304) to achieve a magnetic field in proximity to one or more electrodes, in which the intensity and proximity of the magnetic field are sufficient to immobilize magnetically sensitive beads during the execution of a wash protocol.

Four.

The method of claim 1, wherein step (d) produces a set of drops comprising:

(to)

a drop (1108, 1212) comprising substantially all the beads and having a decreased concentration of the substance with respect to the starting concentration; Y

(b)

 a drop (1110, 1214) that lacks the pearls substantially.

5.

The method of claim 1, wherein the drop (1108, 1212) of step (d) (i) comprises more than 99% of the beads present in the combined drop.

6.

The method of claim 1, wherein the drop (1108, 1212) of step (d) (i) comprises more than 99.999% of the beads present in the combined drop.