RU2380418C1 - Replaceable microfluid module for automated recovery and purification of nucleic acids from biological samples and method for recovery and purification nucleic acids with using thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: replaceable microfluid module for automated recovery and purification of nucleic acids from biological samples contains reagent reservoirs and reaction capacities, microchannels and valves required to handle the liquids in the module. The module comprises an inleak chamber for introduction of the biological sample, a lysis reservoir containing a membrane, reservoirs for dissolved lytic buffers, a microcolumn containing a solid-phase sorbent for binding nucleic acids, a reservoir for flush buffer and ethanol to discharge residual proteins and salt crystals from the microcolumn, an eluting buffer reservoir, an outlet port compatible to a standard microtube, a waste collection reservoir. The method for recovery and purification of nucleic acids from biological samples involves filtration of a sample through the membrane, lysis of the microorganism cells kept on the membrane, binding of nucleic acids on the microcolumn, washing nucleic acids from proteins and salt crystals, elution of nucleic acids from the microcolumn in the tube. All the stages of recovery and purification are carried out in automatic mode in the microfluid module that isolates the sample from environment.

EFFECT: invention allows reducing cost price of the nucleic acid preparation.

14 cl, 17 dwg, 1 tbl, 8 ex

Description

FIELD OF THE INVENTION

The invention relates to molecular biology, microbiology and medicine, and relates to a removable microfluidic module for the isolation and purification of nucleic acids (NK) from biological samples, in which all stages of isolation and purification of nucleic acids, and a method for the isolation and purification of NK from cells of microorganisms are carried out automatically and / or viral particles using a microfluidic module.

State of the art

Analysis of NK is necessary both for conducting a wide range of studies in various fields of science, and for use in clinical diagnostics, pharmacology, medicine, biotechnology, environmental protection, etc. Isolation of NK (DNA and / or RNA), as a rule, is the first stage a number of molecular genetic studies, which also include amplification of NK (PCR and other methods), cloning and sequencing of genome fragments, construction of cDNA libraries, etc. In this regard, obtaining high purity NK preparations remains an urgent task. To isolate NK, both biological material (animal and plant cells, tissues, physiological fluids (blood, saliva, etc.)) and soil, water, and food products are used. Modern requirements for laboratory methods for ND extraction include the universality of the protocol for obtaining purified DNA and RNA from various biological samples, the speed, efficiency, and safety of performing the procedure for personnel. There are increasing requirements for the purity of the preparations obtained and the reproducibility of the procedure.

The following methods are used to concentrate microorganism cells from biological samples: sample filtration (Icuta, K., Maruo, S., Fujisawa, T., Yamada, A. Micro Concentrator with OptoSense Micro Reactor for Biochemical IC Chip Family - 3D Composite Structure and Experimental Verification Proc. Of 12th IEEE International Conference on Micro Electro Mechanical Systems (MEMS'99), Orlando, 1999: p.376-381), electrokinetic focusing (CRCabrera, P. Yager, Continuous concentration of bacteria in a microfluidic flow cell using electrokinetic techniques. Electrophoresis, 2001, v. 22, p. 355-362), microdialysis (F.Xiang, Y. Lin, J.Wen, DWMatson, RDSmith. An integrated microfabricated device for dual microdialysis and on-line ESI -ion trap mass spectrometry for analysis of complex biological samples. Anal Chem., 1999, v. 71, p .1485-1490), affinity chromatography for cell concentration and subsequent solid-state extraction of NK (C. Yu, MHDavey, F. Svec, JM Frechet, Monolithic porous polymer for on-chip solid-phase extraction and preconcentration prepared by photoinitiated in situ polymerization within a microfluidic device. Anal Chem., 2001, v. 73, p. 5088-5096).

Most preferred is the procedure for filtering cells through a specialized membrane due to its simplicity, expressivity and the possibility of implementation in a miniature device. Moreover, concentrated cells can be lysed directly on the membrane after filtration.

Filters made of borosilicate glass, cellulose membranes, PTFE filters, polyvinylidene difluoride membranes can be used as filtration membranes. However, a number of materials is unsuitable for use as filter materials:

- filters made of borosilicate glass fiber may contain a residual amount of water (solutions) after filtration is completed, and also have a strong inhibitory effect against PCR;

- cellulose membranes are fragile and also inhibit PCR;

- PTFE filters, due to their hydrophobic properties, are not suitable for filtering aqueous solutions.

Most preferred are polyvinylidene difluoride filters (PVDF filters). Due to its hydrophilic structure, chemical inertness, mechanical strength, ease of processing and the absence of an inhibitory effect on PCR, this type of filter can be used to perform mechanical concentration and chemical lysis of cells in the form of a miniature closed device (microfluidic module).

The main disadvantage of the known methods of lysis of cells and viral particles is that during the procedure, along with the target nucleic acids, many substances are extracted that are capable of inhibiting PCR.

Among the main methods of physical destruction of cells, such as the freeze-thaw method (CRKuske, KLBanton, DLAdorada, PCStark, KKHill, PJJackson, Small-Scale DNA Sample Preparation Method for Field PCR Detection of Microbial Cells and Spores in Soil . Appl Environ Microbiol, 1998, v. 64, p. 2463-2472), method of suspended homogenization (W. Liesack, E. Stackebrandt, Occurrence of novel groups of the domain Bacteria as revealed by analysis of genetic material isolated from an Australian terrestrial environment. J Bacteriol., 1992, v. 174, p.5072-5078), ultrasonic irradiation method (T. S. Marentis, B. Kusler, GGYaralioglu, S. Liu, EOHaeggstrom, BTKhuri-Yakub. Microfluidic sonicator for real-time disruption of eukaryotic cells and bacterial spores for DNA analysis. Ultrasound Med Biol., 2005, v.31, p. 1265-1277), method ra erases in liquid nitrogen (RAHurt, X. Qiu, L. Wu, Y. Roh, AV Palumbo, JMTiedje, J. Zhou, Simultaneous recovery of RNA and DNA from soils and sediments. Appl Environ Microbiol., 2001, v. 67, p.4495-4503), the most used are methods of freezing-thawing and suspended homogenization. However, such approaches are limitedly implemented in miniature microfluidic systems.

Chemical methods of lysis use various solubilizing and destabilizing agents, such as surfactants, for example SDS (JB Herrick, DNMiller, ELMadsen, WCGiorse, Extraction, purification, and amplification of microbial DNA from sediments and soils. In: JF Burke (ed.), PCR: essential techniques. John Wiley & Sons, NY, 1996, p. 130-133), Triton X-100, sarcosyl et al. (WEHolben, Isolation and purification of bacterial community DNA from environmental samples. In: CJ Hurst, GRKnudsen, MJMcInerney, LD Statzenbach, MV Walter (ed.), Manual of methods in environmental microbiology. American Society for Microbiology, Washington, DC, 1997, p. 431-436), chaotropic agents such as guanidine thiocyanate (hydrochloride) and sodium perchlorate. In addition, chemical lysis includes a stage of high-temperature incubation (60 ° C or more).

A number of lysis procedures are also based on the use of enzymes for the destruction of cell walls and membranes, such as lysozyme, subtilisin, proteinase K (P.A. Rochelle, JCFry, RJParkes, AJWeightman, DNA extraction for 16S rRNA gene analysis to determine genetic diversity in deep sediment communities. FEMS Microbiol. Lett, 1992, v. 100. p. 59-66).

A variety of extraction and purification methods have been developed to isolate NK (see, e.g., J. Sambrook, DW Russell, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, 2001, NY, v. 1, ch. 1, 2, 6 , 7; Short protocols in molecular biology, Wiley, 1995). Well-known methods for the isolation of plasmid and genomic DNA include destruction (lysis) of cells by various methods and subsequent extraction of NK with phenol, a mixture of phenol with chloroform, and ND precipitation with ethanol. CsCl ₂ gradient centrifugation was also used. Chromatographic methods are used to purify nanocrystals. To isolate mRNA from cells and tissues, a sample is extracted with a mixture of phenol - guanidine thiocyanate - chloroform. The disadvantages of such methods are the duration of NC extraction (at least 3.5 h), the multi-stage process, the need to use organic solvents, and the low yield of the product (no more than 50%, for RNA - about 7%).

For the extraction of NK from cell lysates of microorganisms and / or viral particles, gel filtration on Sepharose / Sephadex resins and solid-phase NK extraction on silicon sorbents are used. Solid-phase extraction of ND is the preferred method, since, in addition to the simplicity and speed of the procedure, this principle of nucleic acid isolation is easily adapted to the microfluidic system format.

Currently, the following types of sorbents are used for ND extraction:

- silica gels;

- fiberglass filters;

- chemically modified glass.

A known method of DNA extraction from clinical samples using a silicate sorbent (Short protocols in molecular biology, Wiley, 1995), as well as finely divided (standardized in size) glass (M. Beld, C. Sol, J. Goudsmit, R. Boom, Fractionation of nucleic acids into single-stranded and double-stranded forms. Nucl. Acids Res., 1996, v.24. p.2618-2619). The method consists in adsorption of DNA on a silicate sorbent in the presence of guanidine thiocyanate, followed by washing and elution. The silicate sorbent has a low capacity (per 1 mg of fine glass: 0.5 μg of DNA is adsorbed). This method allows to isolate DNA with a length of more than 40,000 bp, while it is possible to elute from the silicate sorbent no more than 50% of the DNA due to the fact that from 50 to 60% of the adsorbed DNA irreversibly binds to fine glass. Thus, a DNA yield of at least 40,000 bp makes up from 40 to 50%.

Solid-phase DNA purification methods are described that can be carried out on a specialized microchip or implemented in a microfluidic format (Nucleic acid purification using silica gel and glass particles, US Pat. No. 5808041. Methods are based on sorption of DNA on quartz particles or in a sol-gel system in the presence of chaotropic salts, followed by removal of impurities during washing with a water-alcohol mixture and elution of DNA with a buffer solution in which PCR can be performed (K.A. Wolfe, M.C. Breadmore, JPFerrance, MEPower, JFConroy, PMNorris, JPLanders , Toward a microchip-based solid-phase extraction method for isolation of nucleic acids. Electrophoresis, 2002, v.23, p.727-733) Sorbents based on silicon microparticles (sorption in the presence of chaotropic agents) are also used to isolate RNA from biological samples containing DNA and RNA (Process and kit for isolating and purifying RNA from biological sources, US patent 5155018). A known method for the isolation and purification of RNA, including adsorption on Aerosil in a medium of a chaotropic agent, separation of the sorbent and associated NK, elution of NK from Aerosil and purification of the target product. (O.G. Gribanov, A.V. Shcherbakov, N.A. Perevozchikova, V.V. Drygin, A. A. Gusev. A simple method for the isolation and purification of RNA. Bioorg.chemistry, 1997, v.23, p. 763-765). The disadvantages of these methods are the duration, complexity and not high enough yield of the target product (NK) (not more than 50%).

The use of gel filtration (Method for separating long-chain nucleic acids, US Pat. No. 5,057,426) and ion exchange matrices (pH dependent ion exchange matrix and method of use in the isolation of nucleic acids, US Pat. No. 6310199; Method, are also described for the isolation and purification of ND. and system for RNA analysis by matched ion polynucleotide chromatography, U.S. Patent 6,475,388).

Methods are described for separating nanocrystals using metallic magnetic particles, such as particles of iron, zinc, etc., containing carriers that are capable of specifically adsorbing nanocrystals. Magnetic particles with adsorbed nanocrystals are deposited under the influence of a magnetic field. An increase in the sorption capacity of particles is achieved by modifying their surface (Magnetic particles for purifying nucleic acids, U.S. Patent 6,919,444; Particles having a magnetic core and outer glass layer for separating biological material, US 6255477).

Qiagene has patented a method for sequentially isolating DNA and RNA from the same sample (Method for sequentially isolating DNA and RNA from the same nucleic acid-containing sample, WO 2004/108925), including the addition of a chaotropic agent to the sample, precipitation of NK with alcohol, and interaction with a functional NK-binding surface and sequential elution of DNA and RNA. The method is based on the difference in the kinetics of binding and elution of DNA and RNA. The bonding surface may be, for example, glass balls or magnetic particles.

All of the above methods are a series of manual procedures. The stage of DNA purification is necessarily preceded by cell lysis and precipitation of proteins and insoluble particles; therefore, most NK isolation procedures involve several successive centrifugation steps, which leads to an increase in NK isolation time, an increase in the cost of the procedure, and a decrease in NK yield due to losses in the selection of supernatant containing NK, after centrifugation. The main disadvantages of all manual procedures are the complexity and lack of efficiency (insufficiently high yield of ND). In addition, some organic methods use toxic organic solvents (phenol, etc.), which imposes additional requirements on the organization of premises and personnel qualifications.

Currently, a number of companies have developed automated devices that process clinical samples and extract NK from them. The COBAS AmpliPrep system from Roche Diagnostics (USA / Switzerland) is designed to automatically extract DNA and RNA from biological fluids, such as serum and blood plasma, based on technology using magnetic particles. In combination with the COBAS TaqMan® Assays reagent kit, the system performs fast, real-time, automatic PCR with the DNA obtained to detect hepatitis C, hepatitis B and HIV viruses.

Thermo's KingFisher automated device from Thermo Electron (USA) purifies DNA, RNA and proteins based on the movement of magnetic particles. For analysis, samples of physiological fluids (blood, plasma, urine, saliva) with a volume of 20-200 μl are used. The device is designed for simultaneous processing of 24 samples.

Qiagen (Germany) launches QIAsymphony SP for simultaneous automatic purification of NK from 96 samples, for example, for isolation of viral NK or genomic DNA from blood samples. The extraction technology is based on the use of columns with magnetic particles, the surface of which contains a conjugate of nickel with agarose. The system consists of cartridges filled with the necessary reagents, from which solutions and enzymes are dosed to process the clinical sample in the process of ND extraction. Cartridges are automatically opened during the selection process, which minimizes the risk of infection or contamination.

The BioRobot MDx workstation (Qiagen, Germany) includes 8 channels (columns) with replaceable filters. The input and further advancement of the sample is carried out by vacuum pumps; there is no centrifugation stage. The entire procedure is fully computer controlled according to the appropriate protocol for the isolation of genomic DNA from blood or viral DNA and / or RNA from serum and plasma.

The X-tractor GeneTM System workstation (Corbett Technologies, Australia) is a robotic system designed to automatically extract NK from samples up to 200 μl, fully reproducing manual NK extraction.

Bioneer Corp. claimed device for automatic DNA purification (Automatic DNA purification apparatus, WO / 2001/025482), consisting of numerous containers for solutions, channels for the passage of liquids, adjustable valves, a vacuum unit, racks, syringes for accurate quantitative introduction and suction of liquids. The device also reproduces a manual DNA extraction procedure, but in automatic mode.

An automated robot system for identifying biohazard agents is described (Automatic identification of bioagents, WO / 2005/009202), Isis Pharmaceuticals, USA. The system consists of two units separated by an air lock, in the first of which DNA is extracted and purified, and in the second, PCR to identify the agent. To protect against contamination, the pressure in the first block is maintained higher than the external atmospheric pressure, and in the second block it is lower. The system is fully automated.

All currently existing fully automated devices for the preparation of clinical samples are complex designs based on the use of robotics, which determines their bulkiness, high cost (more than 70 thousand US dollars) and the high cost of analysis and operation. These devices mainly reproduce manual methods of ND extraction, but using robots: moving tubes, multichannel dosing of reagents. They are closed systems that prevent operator intervention in the process. During the operation of such systems, constant purchases of reagents of this company, which are intended for this particular device, are necessary.

Due to the above disadvantages of robotic sample processing systems, there is a tendency to move from bulky fully integrated systems to small flexible devices that manipulate the samples to be studied, allowing the user to modify the components of the device and adjust the process according to specific requirements.

There are relatively simple and cheap devices for processing samples containing nanocrystals, which make it possible to fully and partially automate the process. Millipore Corporation has patented a device for separating NK from cells, viral particles, and mycoplasma, consisting of a system of filtering elements (porous membranes) through which a sample is passed sequentially (Filter device for the isolation of a nucleic acid, EP 183242 A2). Cell lysis is performed on a first porous membrane made of polysaccharide or polyethersulfone; the resulting lysate enters a glass fiber filter for subsequent purification and elution of the NK. The filtering membranes are connected by a valve system. The device can be either disposable or reusable. For the simultaneous processing of several samples, several filtering devices can be collected in a cassette. The disadvantages of this device are the need for manual introduction of reagents and the presence of centrifugation stages.

A method for cleaning NK by passing a sample through a system of static mixers on which cell lysis, deposition of cell debris, deposition of NK from a lysate, followed by centrifugation and purification by ion exchange chromatography (Methods for purifying nucleic acids, WO / 2000/005358, Valentis) are described. , USA). The disadvantages of this method, as well as the previous one, are the need for centrifugation.

A device for separating NK (Nucleic acid isolation, WO / 2005/012521, Inivitrogen Corp., USA) is patented, which is a cartridge consisting of a tube connected to a filter element and then to a column for cleaning NK. The filter element contains several layers of filters, the column includes a carrier capable of binding NK, for example a carrier with a variable charge. Sample manipulations are carried out using a syringe or pump. The disadvantage of this method is the need for preliminary processing of the sample before introducing it into the cartridge: cell lysis, dilution of blood serum, etc.

In the last 10 years, devices for processing biological samples and separating nanocrystals based on microfluidic or microfluidic systems containing a network of microchannels with a diameter of 100 μm or less, through which biological samples and reagent solutions are passed, as well as reservoirs in which the components are separated, have become widespread. , concentration and other procedures. Microfluidic systems also include mixing devices, microdosers, micropumps, filters, etc. A number of microfluidic devices are described for producing purified nucleic acids, which are used for various purposes, for example, for PCR. Simple microfluidic devices have been proposed in which NC extraction is carried out in one or several stages, as well as complex computer-controlled systems consisting of numerous microchannels, valves and reservoirs.

Examples of fairly simple devices are a microfluidic device for DNA purification in the interaction of a sample with diatomite (Purification and amplification of nucleic acids in a microfluidic device, WO / 2008/002725, Bio-Rad, USA); a microfluidic device for isolating genomic DNA from human leukocytes, in which DNA from a sample binds to the surface of a channel modified with a DNA binding reagent (DNA purification and analysis on nanoengineered surfaces, WO / 2006/081324); a microchip in which DNA purification occurs by sequentially passing through a series of chromatographic microcolumns that selectively sorb impurities such as proteins, peptides, lipids, pectins, etc., and the last column selectively binds DNA (DNA purification in a multi-stage, multi- phase microchip, WO / 2008/058204). The disadvantages of such devices are the low yield of NK, as well as the need for manual introduction of reagents at different stages of isolation and purification.

A microfluidic device and a method for concentrating and purifying NK from biological samples are described (Methods for nucleic acid isolation and kits using a microfluidic device and concentration step, WO / 2005/068627, 3M Innovative Properties company, USA), including a reservoir for loading a sample and a chamber for processing and mixing, connected by channels and valves. After loading, the sample is subjected to the action of a lysing solution, then passes to the processing chamber, where several stages of concentration and dilution take place, as well as additional lysis and removal of the solution containing inhibitors that interfere with PCR with the obtained DNA. The disadvantage of this system is the possibility of only partial purification of NK, mainly from PCR inhibitors.

A microfluidic cartridge is described for differentiated lysis of various types of cells, including sonication and lysis using chemical agents, and further partial DNA purification (Microfluidic differential extraction cartridge, WO / 2005/028635, Microfluidic systems, USA). This microfluidic is intended mainly for cell lysis, and with its help only partial purification of NK is carried out.

A monolithic microfluidic device for extracting DNA and RNA, mainly from blood samples (Nucleic acid purification chip, WO / 2005/066343, Singapore), consisting of a silicon-based substrate, inlet and outlet openings for introducing and removing a blood sample, and buffers for processing a sample in the amount of several microliters, a micromixer, a lysis chamber, a chamber containing DNA-binding material and a valve system. Also described is a method of manufacturing a monolithic device containing the above components. This device allows you to select NK only from a sample of a certain type (blood).

To protect DNA extracted using microfluidic systems from contamination, a device is proposed for automatic NK isolation from biological samples (Automated nucleic acid isolation, WO / I 997/047967, Sarnoff Corp., France), consisting of a cassette in which the sample is chemically processed, and a removable sealed container for storing received NK.

An example of a more complex microfluidic system is a device that is a collection of modules, in the first of which binding and purification of the target product (ND) takes place, and the second module is the microfluidic device itself, which detects and analyzes the resulting product (Microfluidic devices, WO / 2008/030631 , Microchip biotechnologies, USA). Cell lysis is carried out by ultrasonic treatment in a flow mode. DNA purification is carried out by interaction with magnetic particles containing an affinity carrier. A magnetic field is created by a rotating magnetic block.

Siemens (Germany) proposed a flat cartridge (card) for automatic analysis of DNA or proteins, containing a system of microchannels and microcavities that form containers for the dry reagents contained in them, as well as a method for its manufacture by injection molding (Arrangement for integrated and automated DNA or protein analysis in a single-use cartridge, method for producing such a cartridge and operating method for DNA or protein analysis using such a cartridge, WO / 2006/042838). Dry reagents are introduced into open channels, which are then sealed with a film. The cartridge is a disposable device. The sample is entered into a cartridge ready for analysis, and the results are obtained in a fully automatic mode when the cartridge is placed in a reader.

A number of microfluidic devices are also described, in which DNA is extracted and amplified simultaneously, and in a number of devices, subsequent detection is also performed. An example of such a system is a device for the isolation and amplification of DNA from biological fluids (Micronics, USA) (Method and system for microfluidic manipulation, amplification and analysis of fluids, for example, bacteria assays and antiglobulin testing, WO / 2004/065010, U.S. patent 7416892). The device is a disposable microfluidic map and is intended for the analysis of bacteria in biological fluids and the diagnosis of certain diseases. The card has a built-in filtering membrane on which cells are retained, which are then lysed. A series of solutions is passed through the membrane sequentially: washing solutions, solutions containing enzymes, solutions for amplification and detection. PCR amplification of the obtained DNA is carried out in the appropriate temperature mode, and then the PCR product is washed off the membrane and transferred to the detection element with a transparent window for visual detection.

A microfluidic system (lab-on-a-chip) is described for the detection of certain infectious agents (University of Pennsylvania, USA). The system includes a sample collector (saliva), a disposable plastic cartridge (microfluidic chip) for sample processing, including a system for lysis, extraction of nanocrystals, PCR, labeling of a PCR product, a platform controller for controlling the flow of reagents, temperature, valves, and also a laser scanner for reading results (Z. Chen, MGMauk, J. Wang, WRAbrams, PLCorstjens, RS Niedbala, D. Malamud, HHBau, A microfluidic system for saliva-based detection of infectious diseases. Ann. NYAcad . Sci., 2007, v. 1098, p. 429-436).

Canon U.S. Life Sciences, USA proposes a device for analyzing genomic DNA, consisting of a cartridge in which DNA is extracted, an injector, with which DNA is transferred to a microfluidic chip, including a PCR amplification zone, a detection zone and a DNA analysis zone (Method and molecular diagnostic device for detection, analysis and identification of genomic DNA, WO 2007028084). DNA is isolated in the reaction chamber using magnetic particles or materials that change properties under the influence of an electric charge.

A fully automated portable microfluidic chip for detecting pathogenic microorganisms by real-time PCR (Real-time PCR detection of microorganisms using an integrated microfluidics platform, WO / 2006/085948, Cornell Research Foundation, USA) is proposed. The microchip includes a module for DNA purification, which is connected via microchannels to a module for PCR detection. Cell lysis is carried out in a solution containing chaotropic salts; Further, washing with an aqueous-alcoholic solution is carried out to remove proteins and lipids. The automatic detection system consists of an integrated microprocessor, a system of valves and pumps, devices for thermal cycling and fluorescence detection. Detection is carried out by real-time PCR using a fluorescent dye. The device can be used both in laboratories and in the field.

CombiMatrix Corp., USA, has developed a miniature microfluidic device, consisting of a DNA microchip containing 12000 probes, and a microfluidic cartridge, which includes microfluidic pumps, mixers, valves, microchannels, microreserves for reagents. An electrochemical synthesis of oligonucleotide probes is carried out on a chip. The mixing of liquids in the cartridge is carried out by passing microbubbles generated by electrochemical micropumps. Electrochemical pumps are also used to pump liquids inside the cartridge. After the sample is introduced, PCR and hybridization are performed on a microchip. The device is completely autonomous, i.e. no external sources of reagents, pumps, etc. are required, which eliminates the possibility of sample contamination. The device was used to identify influenza A virus subtypes by genotyping of hemagglutinin and neuraminidase gene regions (RHLiu, MJLodes, T.Nguyen, T. Siuda, M.Slota, HSFuji, A.McShea, Validation of a fully integrated microfluidic array device for influenza A subtype identification and sequencing. Anal. Chem., 2006, v. 78, p. 4184-4193), as well as for studying the expression of human leukemia cell genes (RHLiu, T. Nguyen, K. Schwarzkopf, HSFuji, A. Petrova, T. Siuda, K. Payvan, M. Bizak, D. Danley, A. Mc Shea, Fully integrated miniature device for automated gene expression DNA microarray processing. Anal. Chem., 2006, v. 78, p. 1980-1986 )

An analysis of the scientific and patent literature allows us to conclude that at present there are many methods for the isolation and purification of ND, some of which allow the automation of the process. However, despite the fact that a number of devices have been developed for the automatic production of NK, there is no universal device that allows one to quickly, reproducibly and with high yield separate NK from biological samples in a fully automatic mode. Existing automated robotic systems are expensive, cumbersome and difficult to use, while simpler systems either perform incomplete purification of NK, or NK output is insufficient, or they are designed to isolate DNA from a sample of a certain type, for example, only from blood, saliva, etc., or analysis of the sample for the presence of one specific disease.

In the laboratories of the Russian Federation, at present, all procedures for the isolation and purification of nucleic acids from clinical material are carried out manually. The lack of inexpensive and reliable equipment to automate the processing of clinical samples creates conditions for an increased risk of infection with infectious material, and also leads to false positive and false negative results. The latter inevitably affects the prevention of public health and the effectiveness of treatment of patients.

Thus, in this area there is an urgent need to develop a device for automated extraction of NK, which would be favorably different from the known solutions from the prior art by the simplicity of analysis, the efficiency of isolation (high yield) of NK, as well as low cost.

Disclosure of invention

The first aspect of the invention relates to a replaceable microfluidic module for automated isolation and purification of nucleic acids from biological samples, containing reagent tanks and reaction volumes, microchannels and valves necessary for moving liquids within the module. The microfluidic module includes:

a) a receiving chamber for introducing a sample;

b) a lysis tank containing a membrane for retaining microorganism cells and / or viral particles on it;

c) reservoirs for solutions of lysis buffer containing reagents for the effective destruction of the cell wall and / or viral capsid;

g) a microcolumn containing a solid-phase sorbent for binding nucleic acids;

e) tanks for washing buffer and ethanol for washing the microcolumn in order to remove residual proteins and salt crystals;

e) a reservoir for an elution buffer for dissolving nucleic acids bound on a solid-phase sorbent in a microcolumn;

g) an output port compatible with a standard microtube;

h) a tank for collecting reaction waste.

In one of its embodiments, the replaceable microfluidic module is characterized in that it contains only pneumatic valves for switching gas-liquid flows, while all the necessary electromagnetic components for supplying pressure, heating, mixing in the individual tanks of the module are located in the control unit-controller .

In its next embodiment, the microfluidic module is characterized in that in the lysis tank the module contains a metal rod in an inert shell, necessary for mixing at the stage of lysis.

Finally, in yet another embodiment, the microfluidic module is characterized in that the module is made of materials that are inert with respect to the reagents used and do not sorb NK. Can be used materials, such as, for example, polypropylene, Plexiglas, latex, silicone, but not limited to.

Another aspect of the invention is a method for the automated isolation and purification of nucleic acids from biological samples using a removable microfluidic module containing a set of reagents necessary for the implementation of the procedure for the allocation of NK.

The method is characterized in that all manipulations for the extraction and purification of nanocrystals are performed inside a removable microfluidic module that isolates the sample from the external environment.

The method involves the following successive stages, performed in automatic mode, after entering the test sample into the receiving chamber of the microfluidic module:

a) filtering a biological sample through a membrane that traps cells of microorganisms and / or viruses;

b) lysis of microorganisms and / or viruses detained on the cell membrane;

c) binding of nucleic acids on a microcolumn containing a solid-phase sorbent;

d) washing of nucleic acids from proteins and salt crystals on a solid-phase sorbent column;

e) elution of nucleic acids from a microcolumn into a separate tube.

In one embodiment, the method is characterized in that, in step (a), the biological sample is filtered through a membrane in order to trap microorganisms and viruses on it. The filtration membrane is selected from the group of membranes, including, but not limited to, polyvinylidene difluoride, borosilicate glass, and cellulose membranes.

In its next embodiment, the method is characterized in that the lysis of microorganisms and viruses on the membrane in stage (b) is carried out by sequential treatment with reagents that destroy the cell walls of bacteria and / or the shell of viral particles. As enzymes for the destruction of cell walls and membranes, lysozyme, subtilisin, proteinase K and other enzymes are used. Then add a lysis buffer containing a chaotropic agent in high concentration (3-6 mol / l). Chaotropic reagents are selected from the group of guanidine salts, for example, but not limited to guanidine thiocyanate or guanidine hydrochloride.

In its next embodiment, the method is characterized in that, in step (c), NK is sorbed on a solid-phase sorbent in the presence of a solution of a chaotropic agent in a high concentration (3-6 mol / L) by passing the lysate through a microcolumn containing a solid-phase sorbent. The solid-phase sorbent is selected from the group of sorbents, including silica gels, glass fiber filters, chemically modified glass.

In yet another embodiment, the method is characterized in that, in step (d), washing of the proteins and salt crystals is carried out by sequentially passing through a microcolumn containing NK, solutions of washing buffer and ethanol from the corresponding reservoirs of the removable module, and washing the NK off microcolumns are carried out by passing an eluting buffer supplied from the corresponding reservoir of the microfluidic module through the microcolumn into a separate microtube.

In yet another embodiment, the method is characterized in that steps (a) to (e) are performed sequentially in automatic mode in a replaceable microfluidic module, which is placed in a controller installation controlled by software that implements a control algorithm for valves moving biological sample solutions and reagents in the tanks of the plug-in module.

As a biological sample for the isolation and purification of NK, samples of blood serum, blood plasma or culture fluids containing microorganism cells and / or viral particles can be used.

Other aspects of the present invention will be apparent from the accompanying figures, detailed description, and claims.

The invention is illustrated by the following figures, in which:

Figure 1 is a diagram of the selective extraction of NK from a cell lysate and / or viral particles on a microcolumn containing a solid-phase sorbent.

Figure 2 is a schematic diagram (configuration) of a removable module with the designation of tanks with the necessary reagents, as well as volumes in which the lysis of bacterial cells / virus particles and the extraction of NK.

Designations: K1, K2A, K2B, K3, K4, K4A, K5 - liquid / air valves.

Figure 3 represents the structural elements of the replaceable module. Designations:

1 - the main working platform forming the reservoirs (plexiglass);

2 - elastic membrane (nitrile);

3 - half-shell (plexiglass);

4 - insert channel-forming, self-sealing, with plugs punctured to refuel the module (latex);

5 - top cover (plexiglass);

6 - steel rod in a Teflon shell (mixer);

7 - a sealing ring (latex);

8-membrane (filter) for cell concentration;

9 - cylinder with a suspension of silica gel (microcolumn);

10 - fixing clips.

Fig. 4A is a photograph of a replacement module assembly with charged reagents.

Fig. 4B represents the elements of a replaceable module (working platform and half-housing).

5 is a photograph of a control unit-controller (top view) with a plug-in module connected.

Designations:

1 - electrovalves;

2 - nozzles.

6 is a diagram of the device of pneumatic valves and reservoirs for storing solutions of a removable module for the allocation of NK.

Designations:

1 - a reservoir with a solution;

2 - channels for the flow of solutions;

3 - holes for air supply;

4 - valve;

5 - membrane.

Fig. 7 (A-B) represents the principle of moving solutions from a reservoir to a reservoir through a pneumatic valve in a replaceable module for extracting ND.

Fig. 8 represents the principle of controlling the direction of flow of solutions in the reservoirs of the plug-in module for the allocation of NK.

Designations:

1 - valve 1;

2 - valve 2.

Fig.9 represents the principle of mixing solutions in the reservoir of the replaceable module for the selection of NK.

Figure 10 represents a dialog box of the client part of the software for controlling the process of allocating NK in a plug-in module.

11 represents the result of the isolation of NK from gram-negative bacterial cells (Escherichia coli and Salmonella typhimurium) using an automated isolation method based on a removable microfluidic module and a standard isolation method. Electrophoresis in 1% agarose gel. Tracks:

1 - Molecular marker λ / Hind III;

2 - NK isolated from E. coli culture (~ 10 ⁹ cells / ml) by the standard method;

3 - NK isolated from the culture of S. typhimurium (~ 10 ⁹ cells / ml) in the microfluidic module;

4 - NK isolated from E. coli culture (~ 10 ⁹ cells / ml) in microfluidic module.

Fig. 12 represents the result of NK isolation from gram-positive bacterial cells (Bacillus thuringiensis, ~ 10 ⁹ cells / ml) using an automated isolation method based on a removable microfluidic module and a standard isolation method. Electrophoresis in 1% agarose gel. Tracks:

1 - NC, isolated by the standard method;

2 - NK isolated using a microfluidic module;

3 - NK isolated using a microfluidic module without the addition of lysozyme;

4 - molecular marker λ / Hind III.

Fig. 13 presents the results of PCR with hepatitis B virus DNA as a matrix isolated using a microfluidic module from various dilutions of a blood plasma sample (10 ⁸ -10 ¹ virus particles in the sample initially).

Designations:

'K-' - negative reaction control;

'M' - molecular marker '100 bp Ladder' (Sileks, Russia).

Figa presents the results of PCR with E. coli DNA as a matrix isolated from various dilutions of cells (10 ⁸ -10 ² in 1 ml of the original sample) using an automated procedure based on a removable microfluidic module. Designations:

'K-' - negative reaction control;

'K +' - positive control of the reaction (DNA isolated using the standard method was used as a matrix);

'M' - molecular marker 'PUC / MSP I' (Sileks, Russia).

Fig.14B presents the results of PCR with B. thuringiensis DNA as a matrix isolated from various dilutions of cells (10 ⁸ -10 ² in 1 ml of the original sample) using an automated procedure based on a replaceable microfluidic module.

'K-' - negative reaction control;

'K +' - positive control of the reaction (DNA isolated using the standard method was used as a matrix);

'M' - molecular marker 'PUC / MSP I' (Sileks, Russia).

The implementation of the invention

The aim of the present invention is to provide a removable microfluidic module for the automated isolation and purification of nucleic acids from biological samples and a method for the automated isolation of nucleic acids from biological samples using a microfluidic module.

Replaceable microfluidic module for automated isolation and purification of nucleic acids from biological samples contains:

a) a receiving chamber for introducing a biological sample;

b) a lysis tank containing a membrane for retaining microorganism cells and / or viral particles on it;

c) reservoirs for solutions of lysis buffer containing reagents for the effective destruction of the cell wall and / or viral capsid;

g) a microcolumn containing a solid-phase sorbent for binding nucleic acids;

e) tanks for washing buffer and ethanol for washing the microcolumn in order to remove residual proteins and salt crystals;

e) a reservoir for an elution buffer for dissolving nucleic acids bound on a solid-phase sorbent in a microcolumn;

g) an output port compatible with a standard microtube;

h) a tank for collecting reaction waste.

In the process of ND extraction and purification inside the interchangeable microfluidic module, the biological sample is filtered through the membrane, lyses microorganisms and viruses trapped on the membrane, the lysate is passed through a microcolumn containing a solid-phase sorbent, nucleic acids are bound to the sorbent in the presence of a high concentration of chaotropic agent, followed by washing from proteins and salt crystals and elution of nucleic acids from a microcolumn into a separate tube.

A biological sample is introduced into the receiving chamber of the microfluidic module. The sample solution is then passed through a membrane of polyvinylidene difluoride, borosilicate glass, cellulose or other materials that trap microorganisms and / or viruses. Lysis of microorganisms and viruses detained on the membrane is carried out directly on the membrane.

The process of destruction of the cell wall of microorganisms and / or the viral envelope occurs through a combined chemical and enzymatic lysis, where the cells and / or virus particles detained on the membrane are sequentially treated first with an enzyme solution to destroy the cell walls and membranes (for example, lysozyme), then with a lysis buffer containing chaotropic agent (guanidine thiocyanate at a concentration of 5 mol / l) and detergent (Triton X-100). A chaotropic agent (guanidine thiocyanate) is used to solubilize cell proteins and remove cell wall residues. In addition, in the presence of a chaotropic agent, nucleic acids are easily sorbed on silicate surfaces, which made it possible to remove the stage of extraction of nucleic acids with a phenol-chloroform mixture, as well as to adapt the method to the microfluidic module format.

The destruction of cells followed by solid-phase sorption-desorption allows you to get the yield of NK, equivalent to the standard method using extraction with a mixture of phenol-chloroform and subsequent reprecipitation with ethanol (Examples No. 4-8).

Isolation and purification of nanocrystals using solid-phase sorption are based on their irreversible (relative to dissolution in the same buffer) adsorption on the solid phase; then flushing and elution are carried out. In the process of sorption, the removal of impurities that inhibit enzymatic reactions occurs. Only about 20% of the total surface area of the sorbent particles is available for binding ND to the sorbent structure. However, the rest of the surface, including the inner surface of the pores, remains free to bind small impurities, such as the heme - non-protein part of hemoglobin, a well-known PCR inhibitor. The presence of this additional binding area for fine impurities is very important for the entire column, because it prevents the "competition" of molecules of small impurities and DNA for binding sites.

The principle of the method of separation of NK through sorption-desorption on the sorbent is as follows (Figure 1). NK solution, together with other components of microorganism cells and / or viral particles in a lysis / sorption buffer, is passed through a microcolumn filled with a sorption substance (solid-phase sorbent). Due to the specific composition of the lysis / sorption buffer, nucleic acids bind to the sorbent, while other components (proteins, carbohydrates, polysaccharides, lipids, etc.) are not sorbed. At the end of the sorption, the column is washed with washing buffers containing ethanol, due to which the column is washed from salt crystals and other low molecular weight compounds that are non-specifically adsorbed on the sorbent; due to the insolubility of NK in alcohol, DNA and RNA remain on the column. After drying the column with air flow, NK are eluted with a low salt buffer (TE buffer) or nuclease-free water. Thus, the cleaning of the NK using the described procedure takes no more than 15 minutes.

Thus, the following reagents and buffers are used when separating and purifying NK using a microfluidic module:

at the stage of lysis, a solution of enzymes for the destruction of cell walls and membranes, such as lysozyme, subtilisin, proteinase K, etc., and Lysing buffer (LB) containing a high concentration of a chaotropic agent (3-6 mol / L). As chaotropic agents, for example, guanidine salts (guanidine thiocyanate, guanidine hydrochloride, etc.) can be used;

at the stage of ND extraction in a microcolumn containing a solid-phase carrier (silica gels, glass fiber filters, chemically modified glass, etc.), a washing buffer (PB) containing a chaotropic agent, as well as a solution of 70% ethanol;

at the stage of elution of NK from the sorbent, a solution of the buffer for elution, for example, TE buffer.

In the claimed invention, a replaceable microfluidic module is proposed in which all stages of NC extraction are successively carried out. The module contains tanks, microchannels and valves that move the reaction liquid volumes in the process of separating nanocrystals, and is made of materials inert to the reagents used and not sorbing the nanocrystals.

A schematic diagram of a replaceable microfluidic module is presented in Figure 2.

A biological sample is introduced into the Sample tank, the volume of which is 500 μl. The volume of the reservoir 'Lysozyme' is 100 μl, which is quite enough with the used working concentration of the enzyme. The volume of the reservoir containing Lysis buffer (LB) is 500 μl. The retention of cells and / or viral particles on the membrane is carried out by filtering the sample from the reservoir "Sample" through the reservoir "Filter. Lysis tank 'into the vessel' Reaction waste tank ', the volume of which is 2 ml. Due to the fact that in the vessel 'Filter. The lysis tank 'sequentially treats the cells first with lysozyme, then with lysis buffer, the total volume of this vessel is 700 μl.

Upon reaching the required composition of the lysing mixture, it is passed through a microcolumn containing silica gel as an agent sorbing NK. The volume of this tank is determined by the volume of the suspension of silica gel and is 50 μl. At the design stage, the tank volume for the Wash Buffer (WB) is 500 μl. The volume of the Ethanol reservoir used in the plug-in module is 400 µl. Finally, in order to concentrate the NK preparation sorbed in a microcolumn, the volume of the elution buffer (TE) is 100 μl. Thus, at the design stage of the plug-in module, the total volume of all vessels and microchannels is less than 10 ml.

The temperature optimum of lysozyme is 37 ° C. Accordingly, this temperature is provided in the tanks' Lysozyme 'and' Filter. Lysis reservoir 'when reaching this stage of NK isolation.

The lysing buffer contains a chaotropic agent, for example guanidine thiocyanate (GTZ), in a concentration above 3 mol / L. In this case, the GTZ solution in this concentration is capable of forming crystals at temperatures of 37 ° C and below. This circumstance can impede the movement of solutions through the channels of the plug-in module, as well as reduce the concentration of the chaotropic agent as a whole, negatively affecting the lysis and subsequent sorption of nanocrystals on the microcolumn. In connection with the foregoing, in the vessel 'Filter. The lysis tank 'at the stage of adding Lysis buffer is provided at a temperature of 60 ° C.

For the purpose of effective and uniform lysis, this tank provides for mixing of solutions at the stages of treatment with an enzyme to destroy cell walls and membranes and a lysis buffer. Similar temperature requirements are also applied to the 'Prom. a buffer 'containing a solution which contains guanidine thiocyanate in high concentration. Thermostating of the solutions can be carried out, for example, using Peltier elements located in the control device directly under the indicated reservoirs of the microfluidic module. Stirring in volume 'Filter. The lysis tank 'can be carried out by placing a steel rod in a Teflon shell (mixer) with a diameter not exceeding the diameter of the tank and placing an electromagnetic field source in the control device.

In order to possibly modify the method for separating NK for its use in relation to complex objects for the isolation of NK, such as bacterial spores, a 500 µl reserve vessel ('R' in Fig. 2) is provided in the replaceable module for introducing additional reagents required at the stage of lysis .

Based on the design of the plug-in module carried out above and the adaptation of the ND extraction method, the sequence of operations necessary for carrying out the ND extraction and purification procedure in the removable module using the developed method is proposed. The names of the valves are given in accordance with their designation in figure 2.

1. Entering the biological sample into the sample tank of the plug-in module. All the necessary solutions that are part of the NK Protocol are prepared and filled in the corresponding reservoirs of the replaceable module.

2. Activation of valve k1. Applying external pressure to the Sample tank. Filtration of a biological sample from a 'Sample' vessel through a membrane in a 'Filter' tank. Tank for lysis' into the vessel 'Tank for reaction waste'. Closing valve k1.

3. Activation of the k2a valve. Heating tanks' Lysozyme 'and' Filter. Lysis tank 'up to 37 ° C. Adding an enzyme solution from the Lysozyme reservoir to the cells on the filter in the Filter vessel. Reservoir for lysis'. Turn on the mixing device. Closing valve k2a. Incubation of cells with lysozyme in the vessel 'Filter. Lysis tank for 5 minutes.

4. Activation of the k2b valve. Tank heating 'Filter. Lysis tank 'up to 60 ° C. Adding a solution of LB from the reservoir 'Liz. Buffer (LB) 'to the reaction mixture in the vessel' Filter. Reservoir for lysis'. Closing valve k2b. Incubation of the reaction mixture in the vessel 'Filter. Lysis tank for 5 minutes.

5. Turning off the heating element and the mixing device in the vessel 'Filter. Reservoir for lysis'. Activation of valves k3 and k4. The passage of the reaction mixture from the filter tank. Lysis tank 'through a microcolumn with solid phase sorbent' Microcolumn 'into the vessel' Reaction waste tank '. Closing valves k3 and k4.

6. Tank heating 'Prom. buffer 'up to 55 ° C. Supply of external pressure to the tank 'Prom. buffer'. Activation of valves k4a and k4. The passage of the solution from the tank 'Prom. buffer 'through the microcolumn with the sorbent agent' Microcolumn 'into the vessel' Reaction waste tank '. Closing valves k4a and k4. Switching off heating in the tank 'Prom. buffer'.

7. Supply of external pressure to the Ethanol tank. K4 valve activation. The passage of the solution from the ethanol tank through the microcolumn with the sorbent agent Microcolumn into the vessel for the reaction waste tank. Closing valve k4a.

8. The supply of external pressure to the tank 'TE'. K5 valve activation. The passage of the solution from the reservoir 'TE' through the microcolumn with the sorbent agent 'Microcolumn' into the vessel 'Tank for NK'. Closing valve k5.

9. The proposed algorithm for implementing the method for separating NK in a plug-in module, as well as the requirements for volumes and heating of module elements are used to develop liquid and air valves, create a plug-in module and a control unit-controller, as well as software that manages the procedure for extracting NK.

The invention is further illustrated by examples, which are intended to provide a better understanding of the essence of the claimed invention, but should not be construed as limiting the invention.

Examples

Example 1. The structural elements of the microfluidic module for the selection of the PC and the control unit controller.

Figure 3 presents the structural elements of a removable module for highlighting NK.

Module assembly is carried out in the following order:

1. Install the elastic membrane 2 in the working platform 1.

2. Place agitator 6 in the appropriate reservoir 'Filter. Reservoir for lysis'.

3. Insert the half-shell 3 into the corresponding landing holes of the working platform 1.

4. Firmly pressing the half-shell and the working platform to each other, carry out a preliminary fastening of their fixing clips 10 at corner points.

5. Insert 2 o-rings 7 into the corresponding elements of the half-shell 3.

6. Install a membrane for concentrating cells 8 in the appropriate reservoir 'Filter. Reservoir for lysis'.

7. Install a cylinder with a suspension of silica gel in the tank "Microcolumn".

8. Insert the channel-forming insert with punctured plugs 4 for refueling the reagents in the module into the seat in the top cover 5.

9. Insert the elements collected in paragraph 5.1.8 (the top cover and the channel-forming membrane) into the corresponding seats in the half-shell 3 and fix with clips 10.

As a material for the manufacture of a working platform, half-shell and upper cover, plexiglass was used. The tanks and technological holes are made by drilling and polishing to remove glass dust, as well as possible microcracks and microprotrusions capable of nonspecific sorption of nanocrystals in the presence of a high concentration of a chaotropic agent. Similar requirements were imposed on materials for the manufacture of an elastic membrane and a channel-forming insert. For the manufacture of these elements, latex and nitrile were used, previously tested for the absence of sorbing effects in relation to nanocrystals.

Figure 4 shows photographs of the working layouts of the plug-in module. On figa presents a photograph of the refilled module assembly, ready for analysis. On Figb presents the elements of the module - a working platform 1 and a half-housing 3.

The head instrument consists of a set of electrovalves and nozzles, a microcompressor, heating elements, a power supply, an electronic circuit for connecting the controller to a computer.

Electric valves are designed to regulate the operation of pneumatic valves located in a replaceable module. The electric valve is a direct current solenoid. The solenoid rod closes through the inert membrane the capillary connection of the inlet and outlet. The stem is initially spring-loaded and the valve is closed. When a constant voltage is applied to the electromagnet, the stem is retracted, allowing the air pressure created by the microcompressor to lift the diaphragm and open the valve.

The nozzle block is designed to provide the ability to control the pressure supplied to the air lines of the plug-in module (pressure to lock valve stems and pressure to move fluids). To provide the ability to regulate the pressure in the system, the nozzles were controlled by a constant voltage with pulse-width modulation.

The microcompressor is designed to create an air stream. The microcompressor is a diaphragm or plunger pump capable of creating excess air pressure up to 2 atm. Also, to eliminate cavitation after the compressor, a damper was placed in front of the module.

The heating of the respective tanks was carried out by Peltier elements located under the socket of the installation of the replaceable module in the controller unit. For the general control of Peltier elements, valves and nozzles, a single microprocessor unit has been developed.

Figure 5 presents a photograph (top view) of the installation of the controller. The installation contains 16 electrovalves and 2 nozzles, providing control of pneumatic valves. The replaceable module is placed in a special socket for installing the controller. The module is coupled with the controller installation due to the exact positioning of the stems of the controller nozzles with the receiving holes of the replaceable module. Peltier elements and a magnetic stirrer are located under the socket of the plug-in module. The control and power units, as well as the microcompressor, are remote. The layout is controlled via USB2.0 or IEEE802.3i with the help of developed software.

Example 2. The implementation of the management of the movement of gas-liquid flows, as well as mixing solutions in a removable module for the selection of NK.

In the manufacture of a replaceable microfluidic module, the following principles of control and movement of liquid and air flows were formulated and implemented.

- The driving force for pumping liquids and locking valves (pneumatic valves) is the pressure of compressed air.

- Lack of contact of substances in the tanks, both with the atmosphere and with compressed air, due to the presence of elastic membrane spacers.

At the initial moment, all valves are closed, due to the tension of the valve membrane on its nozzles, because structurally, the tension force of the valve membrane is much greater than the tension force of the membrane of the tank (for example, due to the greater thickness of the valve membrane) (see Fig.6). The pumping of solutions is carried out by applying pressure to the membrane of the reservoir with the solution, while the pressure supply to the corresponding valve through which the fluid flows must not be carried out (Fig. 7, A-B). In the volume under the membrane of the tank from which it is necessary to pump the solution, an excess pressure should be applied that can overcome the valve resistance. The adjustment of the fluid flow rate is carried out by increasing / decreasing the supplied pressure and / or changing the diameter of the hole.

Management of the direction of flow of the solution is carried out according to the following scheme (Fig. 8). When the same pressure is applied to the volume under the membrane of the solution tank and into the volume under the membrane of valve 1, the solution will not flow through valve 1 (valve 1 is completely closed). All solution will flow through the right valve 2, as in this case, the solution needs to overcome only the resistance of the stretched valve membrane.

For mixing in the vessel of the filter module. A lysis tank 'when treated with an enzyme to destroy cell walls and membranes and a lysis buffer, a niche for a magnetic stirrer was constructed at the base of the tank (Figure 9). This solution avoids two critical issues at once - the integrity of the membrane and the absence of additional dead volumes due to the volume of the mixer. The generation of an external alternating electromagnetic field is carried out using a system of active electromagnets mounted in a controller, which are controlled by a microprocessor unit.

Example 3. The control algorithm for valves that carry out the movement of solutions of reagents and biological sample in the tanks of the replaceable module.

Figure 10 presents the interface of the client part of the software (software) that controls the operation of the installation of the controller with a removable module for the allocation of NK.

The client part of the software is intended for manipulating electrovalves (a1-a7) and pneumatic valves (k1-k5) (opening / closing) by clicking on the icon of the corresponding valve, as well as for setting the incubation temperature of the reaction mixture in the corresponding tanks.

Initialization of data exchange via USB2.0 or Ethernet is carried out using the 'Connect' button. A program stop or an emergency interruption is made using the 'STOP!' Button

The opening / closing of electrovalves is realized by applying a voltage of 12 V to the corresponding output of the electrical part of the valve and is controlled by a special microprogram loaded into the microprocessor. Adjustment is made simultaneously through 15 channels, designed to control 14 valves and a mixing device in the volume of the 'Filter. Reservoir for lysis'.

The movement of the reagent solutions and the biological sample in the plug-in module during the NC extraction process is carried out according to the following algorithm.

Stage 1. Entering the biological sample into the receiving chamber (Reservoir 'Sample') of the module.

- click on the 'Connect' icon (initialization of data exchange on I / O ports, resetting all valves to closed state).

Stage 2. Filtration of a biological sample through a filter in a lysis tank.

- open the electrovalve a1.

- open the pneumatic valve k1.

- open the k4 pneumatic valve.

- visually check the flow of the sample in the liquid phase from the tank 'Sample' into the volume 'Filter. Tank for lysis' and further to the volume of 'Tank for waste reaction'. Biological material (cells) remain on the filter.

- close the pneumatic valve k1.

- close the k4 pneumatic valve.

- close the solenoid valve a1.

Stage 3. Processing of biological material (cells / viral particles) with lysozyme.

- set the temperature of the Lysozyme tank to 37 ° С.

- set the tank temperature 'Filter. Tank for lysis' at 37 ° C.

- wait 1 min.

- open the electrovalve a3.

- open the k2a pneumatic valve.

- visually check the flow of the reagent from the Lysozyme reservoir into the Filter volume. Reservoir for lysis'.

- close the k2a pneumatic valve.

- close the solenoid valve a3.

- turn on the mixing device in the filter tank. Reservoir for lysis'.

- turn off the heating of the tank 'Lysozyme.'

- wait 2 minutes

Stage 4. The final lysis of biological material (cells of microorganisms / viral particles) using Lysis buffer.

- set the tank temperature 'Filter. Lysis tank 'at 60 ° C.

- wait 1 min.

- open the electric valve a4.

- open the pneumatic valve k2b.

- visually check the flow of the reagent from the reservoir 'Liz. buffer 'to volume' Filter. Reservoir for lysis'.

- close the pneumatic valve k2b.

- close solenoid valve a4.

- wait 4 minutes

Stage 5. Sorption of nucleic acids on a microcolumn.

- turn off the agitator in the filter tank. Reservoir for lysis'.

- turn off the heating of the tank 'Filter. Reservoir for lysis'.

- open the solenoid valve a2.

- open the k3 pneumatic valve.

- open the k4 pneumatic valve.

- visually check the flow of the sample in the liquid phase from the reservoir 'Filter. Lysis tank 'through the' Microcolumn 'component to the volume' Reaction waste tank '.

- close the k3 pneumatic valve.

- close the k4 pneumatic valve.

- close the solenoid valve a2.

Stage 6. Washing of nucleic acids in a microcolumn using a wash buffer.

- set the tank temperature 'Prom. buffer 'at 55 ° C.

- wait 1 min.

- open the solenoid valve a5.

- open the k4a pneumatic valve.

- open the k4 pneumatic valve.

- visually check the flow of the reagent from the reservoir 'Prom. buffer 'through the component' Microcolumn 'into the volume' Tank for reaction waste '.

- close the k4 pneumatic valve.

- close the solenoid valve a5.

- turn off the tank heating 'Prom. buffer'

Stage 7. Washing of nucleic acids in a microcolumn using Ethanol.

- open the solenoid valve a7.

- open the k4 pneumatic valve.

- - visually check the flow of the reagent from the Ethanol tank through the Microcolumn component to the volume of the Reaction Waste Tank.

- close the k4 pneumatic valve.

- close the solenoid valve a7.

Stage 8. Elution of the NK from the microcolumn using the TE buffer.

- open the solenoid valve a6.

- open the k5 pneumatic valve.

- visually check the flow of the reagent from the TE tank through the Microcolumn component to the volume of the Tank for NK preparation.

- close the k5 pneumatic valve.

- close the solenoid valve a6.

Stage 9. Resetting the program parameters to the initial state

- click on the 'STOP!' icon

- disconnect from the plug-in module a microtube with a solution of extracted NK.

Example 4. The selection of NK from gram-negative cells using a microfluidic module.

The replacement microfluidic module assembly was carried out as described in Example 1. The reagent and buffer solutions were filled into the corresponding replacement module reservoirs in the following compositions:

Lysing buffer (LB): 5 mol / L GTZ (Sigma, USA), 0.15 mol / L Tris, 0.05 mol / L EDTA and 4.9% Triton X-100 (Sigma, USA), deionized water.

Wash buffer (PB): 5 mol / L GTZ (Sigma, USA), 0.15 mol / L Tris, 0.05 mol / L EDTA, deionized water.

Lysozyme solution (Lysozyme): 10 mg / ml lysozyme, 0.15 mol / L Tris pH 8.0, 0.05 mol / L EDTA, deionized water.

Ethanol: 70% ethanol (rectified).

Elution buffer (TE): 0.15 mol / L Tris pH 7.0, 0.05 mol / L EDTA, deionized water.

In order to prepare the microcolumns of the plug-in module, 8 μl of an aqueous suspension of silica gel G60 Merck (Germany) was applied into a polypropylene cylinder 8 mm high, 1.5 mm in diameter, 10 μl in volume.

0.5 ml of a culture of gram-negative cells of Escherichia coli and Salmonella typhimurium was added to the receiving chamber of the plug-in module, after which the chamber was closed, the module was connected to the controller, the software was started, and NK was isolated according to the algorithm described in Example 3.

As a comparison method, the standard lysis procedure was used, followed by phenol-chloroform extraction and reprecipitation of NC with ethanol (centrifugation at 13 thousand rpm, the total time of the procedure was more than 3 hours). Assessment of the efficiency of NK extraction was carried out according to the electrophoretic picture in a 1% agarose gel, by visual comparison of the intensity of the bands in tracks containing NK isolated using a replaceable microfluidic module and using the standard isolation method

The results of the selection of NK from gram-negative cells using a removable microfluidic module and a standard method of isolation are presented in Fig.11.

As evidenced by the results presented in Fig. 11, the use of an automated procedure based on the microfluidic module allows efficient isolation of both DNA and RNA from gram-negative cells.

Example 5. The selection of NK from gram-positive cells using a microfluidic module.

The replacement microfluidic module assembly was carried out as described in Example 1. The reagent and buffer solutions were filled into the corresponding replacement module reservoirs in the following compositions:

Lysing buffer (LB): 5 mol / L GTZ (Sigma, USA), 0.15 mol / L Tris, 0.05 mol / L EDTA and 4.9% Triton X-100 (Sigma, USA), deionized water.

Wash buffer (PB): 5 mol / L GTZ (Sigma, USA), 0.15 mol / L Tris, 0.05 mol / L EDTA, deionized water.

Lysozyme solution (Lysozyme): 10 mg / ml lysozyme, 0.15 mol / L Tris pH 8.0, 0.05 mol / L EDTA, deionized water.

Ethanol: 70% ethanol (rectified).

Elution buffer (TE): 0.15 mol / L Tris pH 7.0, 0.05 mol / L EDTA, deionized water.

In order to prepare the microcolumns of the plug-in module, 8 μl of an aqueous suspension of silica gel G60 Merck (Germany) was applied into a polypropylene cylinder 8 mm high, 1.5 mm in diameter, 10 μl in volume.

Instead of a lysozyme solution, TE buffer was added to one of the plug-in modules in the Lysozyme reservoir.

0.5 ml of a culture of gram-positive cells of Bacillus thuringiensis was added to the receiving chamber of the plug-in module, after which the chamber was closed, the module was connected to the controller installation, the software was started and NK was isolated according to the algorithm described in Example 3. Isolation was carried out in a plug-in module containing lysozyme solution in an appropriate tank, and in a module containing TE buffer instead of lysozyme in the appropriate volume.

As a comparison method, the standard lysis procedure was used, followed by phenol-chloroform extraction and reprecipitation of NC with ethanol (centrifugation at 13 thousand rpm, the total time of the procedure was more than 3 hours). Assessment of the efficiency of NK extraction was carried out according to the electrophoretic picture in a 1% agarose gel, by visual comparison of the intensity of the bands in tracks containing NK isolated using a replaceable microfluidic module and using the standard isolation method.

The results of the selection of NK from B. thuringiensis cells using a replaceable microfluidic module and a standard isolation method are presented in Fig. 12.

As shown by the results presented in Fig. 12, the addition of lysozyme dramatically affects the efficiency of isolation of both RNA and DNA. In the absence of lysozyme (Lane 3) in the composition of the reagents for lysis in the replacement module, only partial lysis of bacteria is observed, which leads to an almost complete loss of NK. Thus, the stage of processing cells with lysozyme is a necessary component of an automated procedure using a removable module. The developed procedure allows NK to be isolated from gram-positive cells with an efficiency comparable to the standard isolation method.

Example 6. Evaluation of the effectiveness of the isolation of viral DNA from blood plasma samples using an automated procedure based on a removable microfluidic module.

The replacement microfluidic module assembly was carried out as described in Example 1. The reagent and buffer solutions were filled into the corresponding replacement module reservoirs in the following compositions:

Lysing buffer (LB): 5 mol / L GTZ (Sigma, USA), 0.15 mol / L Tris, 0.05 mol / L EDTA and 4.9% Triton X-100 (Sigma, USA), deionized water.

Wash buffer (PB): 5 mol / L GTZ (Sigma, USA), 0.15 mol / L Tris, 0.05 mol / L EDTA, deionized water.

Lysozyme solution (Lysozyme): 10 mg / ml lysozyme, 0.15 mol / L Tris pH 8.0, 0.05 mol / L EDTA, deionized water.

Ethanol: 70% ethanol (rectified).

Elution buffer (TE): 0.15 mol / L Tris pH 7.0, 0.05 mol / L EDTA, deionized water.

In order to prepare the microcolumns of the plug-in module, 8 μl of an aqueous suspension of silica gel G60 Merck (Germany) was applied into a polypropylene cylinder 8 mm high, 1.5 mm in diameter, 10 μl in volume.

Samples of blood plasma (200 μl) containing hepatitis B virus in various concentrations were added to the receiving chamber of the replaceable module, after which the chamber was closed, the module was connected to the controller unit, the software was started, and NK was isolated according to the algorithm described in Example 3.

As a standard technique, viral DNA was isolated from blood plasma using a commercial QIAamp DSP Virus Kit (Cat No. 60704, Qiagen, Germany).

In order to evaluate the effectiveness of the procedure, PCR was performed with isolated DNA as a template and with primers specific for the hepatitis B virus genome (gene X). The results are presented in Fig.13.

As the results shown in Fig. 13 show, the developed automated procedure based on the microfluidic module allows efficient isolation of viral DNA from blood plasma samples of up to 10 ² particles in the test sample. In this case, the obtained DNA preparation can be used directly in PCR.

Example 7. Evaluation of the effectiveness of the selection of NK from bacterial cells (E. coli and B. thuringiensis) in the range of 10 ² -10 ⁸ cells in 1 ml of the original sample using an automated procedure based on a removable microfluidic module.

Replacement microfluidic modules were assembled as described in Example 1. The reagent and buffer solutions were filled into the corresponding interchangeable module tanks in the following compositions:

Lysing buffer (LB): 5 mol / L GTZ (Sigma, USA), 0.15 mol / L Tris, 0.05 mol / L EDTA and 4.9% Triton X-100 (Sigma, USA), deionized water.

Wash buffer (PB): 5 mol / L GTZ (Sigma, USA), 0.15 mol / L Tris, 0.05 mol / L EDTA, deionized water.

Lysozyme solution (Lysozyme): 10 mg / ml lysozyme, 0.15 mol / L Tris pH 8.0, 0.05 mol / L EDTA, deionized water.

Ethanol: 70% ethanol (rectified).

Elution buffer (TE): 0.15 mol / L Tris pH 7.0, 0.05 mol / L EDTA, deionized water.

In order to prepare the microcolumns of the plug-in module, 8 μl of an aqueous suspension of silica gel G60 Merck (Germany) was applied into a polypropylene cylinder 8 mm high, 1.5 mm in diameter, 10 μl in volume.

0.5 ml of a culture of E. coli and B. thuringiensis in dilutions in the range of 10 ² -10 ⁸ cells in 1 ml was added to the receiving chamber of the plug-in module, after which the chamber was closed, the module was connected to the controller-installation, the software was started and isolation was performed NK according to the algorithm described in Example 3.

As a comparison method, the standard lysis procedure was used, followed by phenol-chloroform extraction and reprecipitation of NC with ethanol (centrifugation at 13 thousand rpm, the total time of the procedure was more than 3 hours). The efficiency of NK isolation was assessed by PCR with the extracted DNA as a template and with primers specific for the E. coli and B. thuringiensis genomes (gyrA gene), followed by electrophoretic separation of PCR products on a 2% agarose gel.

On Fig presents the results of PCR with DNA isolated using an automated procedure based on replaceable microfluidic modules from dilutions of cultures of E. coli (A) and B.thuringiensis (B) in the range of 10 ² -10 ⁸ cells in 1 ml of the original sample . As evidenced by the presented results, the inventive method for the automated extraction of NK using replaceable microfluidic modules allows analysis in a wide range of concentrations of bacterial cells in a biological sample.

Example 8. Evaluation of the total losses in the allocation of NK using an automated procedure based on a removable microfluidic module.

The preparation of plug-in modules and the selection of NK from cell cultures of E. coli and B. thuringiensis was carried out as described in Examples 3-7.

The total losses during the extraction of NK at the stage of laboratory testing were estimated by carrying out the stages of NK isolation using an automated procedure based on replaceable microfluidic modules and a standard technique for NK isolation with subsequent measurement of DNA concentrations on a spectrophotometer. The isolation procedure was performed in triplicate. The results are presented in Table 1.

Table 1 Determination of DNA concentrations isolated from E. coli and B.thuringiensis cells using an automated procedure based on microfluidic modules and a standard isolation procedure Concentration of DNA isolated from E. coli cells by standard method Concentrations of DNA isolated from E. coli cells using a microfluidic module Concentrations of DNA isolated from B.thuringiensis cells by standard method The concentration of DNA isolated from B.thuringiensis cells using microfluidic module 12.2 ng / μl 10.8 ± 0.4 ng / μl 14.1 ng / μl 13.6 ± 0.5 ng / μl 1.01 ng / μl 0.9 ± 0.1 ng / μl 1.01 ng / μl 0.866 ± 0.05 ng / μl

The results presented in Table 1 indicate that in all experiments the concentration of nucleic acids isolated using the claimed automated NK procedure, measured by absorption on a spectrophotometer, differed from the concentration of NK isolated by the standard method by no more than 20%, which demonstrates the high efficiency of the developed automated procedure.

Thus, the presented invention allows the isolation and purification of nucleic acids of bacteria and / or viruses from biological samples (blood, plasma, culture fluids) using a removable microfluidic module, in which all stages of the isolation and purification of nucleic acids are carried out automatically. The method includes the stage of concentration (retention) of pathogen cells on the membrane, lysis and subsequent extraction of NK by passing the resulting lysate through a solid-phase carrier with simultaneous sorption of NK on it, washing the sorbent and associated NK, and eluting NK. The invention allows the selection of nucleic acids of cells of microorganisms and / or viruses from biological samples in automatic mode with low losses (not more than 20%), low cost, short time required to obtain a result. The method does not require expensive equipment and highly qualified personnel. The resulting NK preparation can be used without additional purification directly in the amplification or hybridization of NK to directly identify the infectious agent in the test sample or to conduct further molecular genetic analysis.

All patents, publications, scientific articles, and other documents and materials cited or referenced herein are hereby incorporated by reference to the extent that each of these documents was individually incorporated by reference or is presented in its entirety.

Although the preferred embodiments of the present invention and their advantages have been described in detail above, a person skilled in the art will be able to make various changes, additions, without going beyond the scope of this invention, which are defined by the appended claims.

Claims (14)

1. A replaceable microfluidic module for the automated isolation and purification of nucleic acids from biological samples, containing tanks for reagents and reaction volumes, microchannels and valves necessary for moving liquids inside the module, characterized in that it includes:
a) a receiving chamber for introducing a biological sample;
b) a lysis tank containing a membrane for retaining microorganism cells and / or viral particles on it;
c) reservoirs for solutions of lysing buffer containing reagents for the destruction of the cell wall of microorganisms and / or viral capsid;
g) a microcolumn containing a solid-phase sorbent for binding nucleic acids;
e) tanks for washing buffer and ethanol to remove residual proteins and salt crystals from the microcolumn;
e) a reservoir for an elution buffer for dissolving nucleic acids bound on a solid-phase sorbent in a microcolumn;
g) an output port compatible with a standard microtube;
h) a tank for collecting reaction waste. 2. The microfluidic module according to claim 1, characterized in that it contains only pneumatic valves for switching gas-liquid flows, while all the necessary electromagnetic components for supplying pressure, heating, mixing in the individual tanks of the module are located in the control unit the controller. 3. The microfluidic module according to claim 1, characterized in that the lysis tank contains a metal rod in an inert shell for mixing the reaction mixture at the stage of lysis. 4. The microfluidic module according to claim 1, characterized in that it is made of materials inert to the reagents used and not sorbing nucleic acids. 5. A method for automated isolation and purification of nucleic acids from biological samples, comprising the following stages:
a) filtering the biological sample through a membrane that traps cells of microorganisms and / or viruses;
b) lysis of microorganisms and / or viruses detained on the cell membrane;
c) binding of nucleic acids on a microcolumn containing a solid-phase sorbent;
d) washing the nucleic acids from proteins and salt crystals on a solid-phase sorbent column;
e) elution of nucleic acids from the microcolumn into a separate tube;
and characterized in that the biological sample is introduced into the receiving chamber of the microfluidic module according to claim 1, and all stages of the isolation and purification of nucleic acids are carried out inside the module. 6. The method according to claim 5, characterized in that at the stage (b) the lysis of microorganisms and viruses detained on the membrane membrane is carried out by the sequential addition of lysis solutions to destroy the cell wall of microorganisms and / or the viral capsid from the corresponding microfluidic module reservoirs. 7. The method according to claim 5, characterized in that at the stage (c) the binding of nucleic acids on a solid-phase sorbent is carried out by passing the lysate through a microcolumn containing a suspension of a solid-phase sorbent. 8. The method according to claim 5, characterized in that in step (d), the washing from proteins and salt crystals is carried out by sequentially passing through a microcolumn containing nucleic acids bound to a solid-phase sorbent, wash buffer and ethanol solutions from the corresponding replaceable module tanks, and flushing of nucleic acids from the microcolumn into a separate microtube in stage (e) is carried out by passing through the microcolumn an elution buffer supplied from the corresponding microfluidic module reservoir. 9. The method according to claim 5, characterized in that the interchangeable microfluidic module is placed in a controller installation controlled by software that implements an algorithm for controlling valves moving a biological sample and reagent solutions inside the interchangeable module. 10. The method according to claim 5, characterized in that as a biological sample use blood, blood serum, blood plasma or culture fluids containing microorganism cells and / or viral particles. 11. The method according to claim 5, characterized in that they use a membrane to trap microorganisms and / or viral particles made of a material selected from the group consisting of polyvinylidene difluoride, borosilicate glass, cellulose. 12. The method according to claim 6, characterized in that as lyse buffers use a solution of lysozyme and a solution containing a chaotropic agent. 13. The method according to p. 12, characterized in that as a chaotropic agent using guanidine thiocyanate or guanidine hydrochloride. 14. The method according to claim 7, characterized in that the solid-phase sorbent is selected from the group of sorbents, including silica gels, glass fiber filters, chemically modified glass.

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