ISSN 00036838, Applied Biochemistry and Microbiology, 2011, Vol. 47, No. 2, pp. 211–220. © Pleiades Publishing, Inc., 2011. Original Russian Text © D.D. Mamaev, D.A. Khodakov, E.I. Dementieva, I.V. Filatov, D.A. Yurasov, A.I. Cherepanov, V.A. Vasiliskov, O.V. Smoldovskaya, D.V. Zimenkov, D.A. Gryadunov, V.M. Mikhailovich, A.S. Zasedatelev, 2011, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2011, Vol. 47, No. 2, pp. 231–240.
Method for Automated Extraction and Purification of Nucleic Acids and Its Implementation in Microfluidic System D. D. Mamaev, D. A. Khodakov, E. I. Dementieva, I. V. Filatov, D. A. Yurasov, A. I. Cherepanov, V. A. Vasiliskov, O. V. Smoldovskaya, D. V. Zimenkov, D.A. Gryadunov, V. M. Mikhailovich, and A. S. Zasedatelev Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia email:
[email protected] Received June 5, 2010
Abstract—A method and a microfluidic device for automated extraction and purification of nucleic acids from biological samples have been developed. The method involves disruption of bacterial cells and/or viral particles by combining enzymatic and chemical lysis procedures followed by solidphase sorbent extraction and purification of nucleic acids. The procedure is carried out in an automated mode in a microfluidic mod ule isolated from the outside environment, which minimizes contact of the researcher with potentially infec tious samples and, consequently, decreases the risk of laboratoryacquired infections. The module includes reservoirs with lyophilized components for lysis and washing buffers; a microcolumn with a solidphase sor bent; reservoirs containing water, ethanol, and waterethanol mixtures for dissolving freezedried buffer com ponents, washing the microcolumn, and eluting of nucleic acids; and microchannels and valves needed for directing fluids inside the module. The microfluidic module is placed into the control unit that delivers pres sure, executes heating, mixing of reagents, and movement of solutions within the microfluidic module. The microfluidic system performs extraction and purification of nucleic acids with high efficiency in 40 min, and nucleic acids extracted can be directly used in PCR reaction and microarray assays. Keywords: nucleic acids, disposable microfluidic module, device for automated extraction and purification of nucleic acids, biochips. DOI: 10.1134/S0003683811020128
The overwhelming majority of methods currently used in molecular biology are based on specific nucleic acid sequences (NA). The initial step of genebased assays involves extraction of nucleic acids (DNA and/or RNA) from biological samples such as animal and plant cells; tissues and physiological fluids (blood, saliva, etc.); and also from soil, water, and food sam ples. In the context of intensive development of gene based techniques, presentday requirements for labo ratory nucleic acid extraction methods have been changed. More attention has been paid to the poten tial for the automation and application of universal common protocols for extraction and purification of NA. The crucial requirements are the duration, labor intensity of sample processing, simultaneous treat ment of several samples, the quality, yield of target NA, and the safety of performing the technique by staff members of medial and research laboratories. Until recently, extraction and purification of NA from clinical samples are primarily performed manu ally. The limitation of this approach is that it is labor intensive, prone to crosscontamination between sam ples to be tested, and it also involves a risk of infection
for staff members when working with pathogens. For this reason, there is an acute need for systems capable of automated extraction and purification of NA, meeting the presentday requirements. Traditional laboratory techniques for extracting DNA and RNA have been described, for example, in [1–3]. Sample processing for NA extraction involves disruption of cells (membranes, cell walls, or viral capsids), denaturation of nucleoprotein complexes, and removal of proteins and other impurities. Solid phase extraction methods have been widely accepted, which include ionexchange resins, silicon matrices, silica gels, quartz and glass particles and fibers, diato mite, zeolite, etc. [4]. Solid carriers allow extraction and purification of NA to be fully or in part auto mated, for example, glass or quartz particles, solgel system [5, 6], and also such carriers as porous mono lithic matrices based on silicon and various polymers with wide surface area and controlled pore size [7, 8]. Recently, magnetic particles, magnetic micro spheres with surfaceimmobilized ligands for binding biomolecules, have become the most widely used method of extraction and purification of NA from bio
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logical samples [9–12]. Magnetic particles with adsorbed nucleic acids, are precipitated in the mag netic field. Magnetic particles are produced from iron oxide, zirconium, various polymers, porous glasses, etc. An increase in sorption capacity of particles is achieved through modifying their surface. Miniature devices, including all necessary compo nents for carrying out sufficiently complex biochemi cal tests, are increasingly used. These are so called micro total analysis systems, μTAS, microfluidic devices in different formats: cassettes, microarrays, minidiscs, etc; and labonachip and labonaCD [13–16]. Microfluidic systems contain a network of microchannels with a diameter of 100 μm or less. Samples to be tested and reagent solutions are driven through these microchannels and reservoirs in which separation of components, purification, concentra tion, and other procedures occur. Microfluidic sys tems also include stirring devices, pipettes, pumps, fil ters, etc. Experimental microfluidic systems coupling extraction and amplification of DNA have been reported. Some systems are able to detect amplified DNA [17–19]. A number of large foreign companies have devel oped and made commercially available complex work stations for extraction and purification of DNA from biological samples in an automated mode such as KingFisher (Thermo Electron, United States), MagNA Pure LC and Cobas (Roche Diagnostics, Germany), BioRobot and QIAsymphony (Qiagen, Germany), Maxwell System (Promega, United States), and Quickgene (FujiFilm, Japan). Most workstations employ sorption of nucleic acids on mag netic microspheres and tend to copy the protocol of manual extraction of DNA and RNA, including suc cessive movements of tubes and multichannel pipet ting of reagents using robots [20]. Such systems cost several million rubles and the net cost of sample pro cessing remains high enough. Despite the development of Russia’s market of molecular diagnostics, domestic analogues of auto mated systems for NA extraction are completely unavailable. The objective of this work is to create a unified pro tocol for automated extraction and purification of NA from eukaryotic cells, bacteria, and viruses and to develop a device based on unique microfluidic mod ules that would compare favorably with the currently available analogues in terms of simplicity, safety of testing, high yield of target products, and low cost. METHODS Reagents and enzymes were lysozyme; guanidine hydrochloride; guanidine thiocyanate; sodium Nlau ryl sarcosinate; Triton Х100; EDTA; ТЕbuffers;
nucleasefree water (Sigma, United States); protein ase K (Ambion, United States); GeneRulerTM High Range DNA Ladder (Fermentas, Lithuania) and PUC 19/MSPI (Sileks, Russia); RNeasy Mini RNA extraction kits (Qiagen, Germany) and Genomic DNA Purification Kit (Fermentas, Lithuania); ToTALLY RNA (Ambion, United States); glass microfiber filters GF: GF/B, GF/C, GF/D (What man, Germany;), silica gel G60 (Меrck, Germany); and thermostable HotTaq DNApolymerase (Sileks, Russia), a kit for carrying out reverse transcription combined with PCR, “Оnеstep RTPCR kit” (Qiagen, Germany). Oligonucleotides. Oligonucleotides were synthe sized on an ABI394 DNA/RNA synthesizer (Applied Biosystems, United States) using the standard phos phoramidite method and purified with reversedphase HPLC (Gilson, France). Oligonucleotides for immo bilization onto biochip gel elements contained a spacer with a free amino group introduced during the synthesis by 5'AminoModifier C6 (Glen Research, United States). Hydrogel biochips. Biochips were produced as pre viously described [21]. The polymerization mixture containing gelforming monomers and oligonucle otide probes was spotted onto a plastic surface using a robotic Arrayer to form hemispherical drops 600 μm in diameter spaced one from another at a distance of 300 μm. Selection of solidphase carriers and conditions for NA extraction. Optimization of the protocol of extrac tion and purification of NA involved the fabrication of microcolumns into which solidphase carriers were loaded: water suspension of silicagel G60 (5 μl) was loaded into a polypropylene cylinder of 8 mm in height, 1.5 mm in diameter, or two spacers of glass microfiber filters GF (7.0 mm in diameter) were loaded into polypropylene cylinders of 8 mm in height. After lysis, a biologic sample (100–500 μl) was mixed with ethanol. The resulting mix was added into the column with a sorbent. The column was washed with a mixture of 50% ethanol and 0.15 M trisHCl buffer, pH 8.0, containing 0.05 M EDTА, 3 M guani dine hydrochloride, and then with 80% ethanol. The column was allowed to air dry, and nucleic acids were eluted off the column with TEbuffer (10 mM tris HCl, pH 7.5, 1 mM EDTA, pH 8.0) or water. Disposable microfluidic modules for extraction and purification of NA (Figs. 1, 2). The main working plat form, as well as the upper and lower lids of each mod ule, was manufactured by casting inert polymer mate rials that do not adsorb nucleic acids such as polypro pylene, polystyrene, and polymethylmethacrylate (organic glass). The spacers were prepared from elastic materials such as silicon and polyethylene. In this work, we used polypropylene microfluidic modules:
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Fig. 2. (a, b, c) Structural components of a disposable microfluidic module. a—the upper panel (lid, top view), polypropylene, 2 mm. The upper panel includes: 1— openings for solenoid rods (16 units); 2—an opening for the sample inlet chamber; 3—openings for fixing screws; L1, L3—spacers, silicon, 2 mm; L2—spacer, polyethyl ene, 50 μm; b—the main working platform, polypropylene, 20 mm; c—the lower panel (lid), polypropylene, 7 mm.
the main working platform was 20 mm thick, the upper and lower lids were 7 mm thick with silicon spacers Pentelast Т4 (ООО PentaYunior, Russia) 2 mm thick, and a polyethylene film was 50 μm thick. Mod ule elements were fastened with screws.
The preparation of the microfluidic module for work. A lyophilized mix of reagents for the first lysis buffer was loaded into reservoir 2 of the microfluidic module with a volume of 1.0 ml (Fig. 1b), a lyophilized mix of reagents for the second lysis buffer was added into
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Fig. 3. The control unit of the device for automated extraction and purification of NA (a—without a microfluidic module; b—ready for work device with mounted microfluidic module): 1—a unit of solenoids for controlling microfluidic module valves; 2—solenoid rods; 3—a unit of heaters; 4—positioning rails for mounting a microfluidic module into the control unit; 5—microfluidic module; 6—a unit of electromagnetic stirrers; 7—compressor; 8—electronic control group.
reservoir 3 with a volume of 1.5 ml; a lyophilized mix of reagents for the washing buffer with a chaotropic agent was put into reservoir 5 with a volume of 1.0 ml. A water suspension of silicagel G60 (5 μl) was placed into the microcolumn (solidphase sorbent for binding NA) that was installed in corresponding reservoir 8. Res ervoir 4 with a volume of 0.5 ml was filled with 96% ethanol; reservoir 6 with a volume of 0.5 ml was loaded with a mixture of water and ethanol (1 : 1); reservoir 7 with a volume of 1.0 ml was loaded with 80% ethanol; reservoir 9 with a volume of 0.2 ml was loaded with water (or lowsalt buffer, for example, TE buffer). The volume of the waste reservoir was 5.0 ml. After dilution of lyophilized components, the buff ers had the following compositions: 10 mM trisHCl; 1 mM EDTA; 50 mg/ml lysozyme, pH 8.0, (the first lysis buffer); 10 mM trisНC1; 1 mM EDTA; 4.5 M guanidine hydrochloride; 1 mg/ml proteinase K; 0.5% sodium Nlauryl sarcosinate, pH 6.5 (the second lysis buffer); 0.15 M trisНC1; 0.05 М EDTA; 3 M guani dine hydrochloride, pH 8.0 (washing buffer). To stir solutions and reaction mixtures during sam ple processing, stir bars, tefloncoated steel sticks, were placed into reservoirs 2, 3, and 5.
The device for extraction and purification of NA. The device consisted of a microfluidic module, a con trol unit, and a computer with appropriate software to control extraction and purification of NA (Fig. 3). The microfluidic module with all necessary reagents and components for biological sample processing was mounted into the control unit. The control unit included solenoids to control module valves, solenoid rods, a unit of heaters, a unit of electromagnetic stir rers, a compressor, and an electronic control group. To deliver pressure, a diaphragm pump or plunger pump was used, capable of delivering excess pressure up to 2 atm. As electromagnets that control valves, lin ear solenoids хЕРМ were used (Saia Burgess, Hong Kong). The speed of flow regulation was controlled by fluid flow regulator FRDR150 with makeandbreak joints FCDR200…500 (built in) (Atoll, United States). Onestage Peltier modules were used as heat ing elements (ZAO SKTB NORD, Russia) sized 15 × 15 mm. The temperature controller was a circuit based on a TRK02 transducer (ООО TSF, Russia). Extraction and purification of NA was performed from cultures of grampositive Bacillus thuringiensis sp. Sotto TO 4001 cells, gramnegative Escherichia coli K12, and E. coli K12 cells infected with MS2 or λ bac
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Fig. 4. Results of multiplex PCR on a biochip using bacterial and phage nucleic acids extracted in microfluidic modules. (a) Scheme of a biochip for PCR. (b–e) Fluorescent patterns of biochips and histograms of normalized signals of the right and left columns of biochip elements (circled with a dashed line) containing immobilized probes, after carrying out PCR with NA isolated (b) from E. coli cells; (c) from B. thuringiensis; (d) from E. coli cells infected with λ phage; (e) from E. coli cells infected with MS2 phage. In the histograms, background Iref value is shown with a solid line.
teriophages. Cells (0.1–0.5 ml) were placed into the inlet chamber of a disposable module, the module was installed in the control unit, then the software was started, and NA extraction ran in an automated mode. The total extraction time was less than 40 min. As a comparison method, we used a conventional procedure for extraction and purification of DNA including lysis, phenolchloroform extraction, and reprecipitation of DNA with ethanol (centrifugation at 18000 g). NA were also extracted following the pro tocols of manufacturers using the following kits: Genomic DNA Purification Kit (Fermentas, Lithua nia), ToTALLY RNA (Ambion, United States), or RNeasy Mini (Qiagen, Germany). Extraction efficiency of NA was evaluated using electrophoresis on a 1 or 2% agarose gel by visual inspection of band intensity in lines with nucleic acids extracted by the standard methods (manually) and in an automated mode on a disposable microfluidic module. The concentration of extracted DNA was determined by absorption in the UV region at 220 to 320 nm using a Genesys 10 uv spectrophotometer (Thermo Scientific, United States). APPLIED BIOCHEMISTRY AND MICROBIOLOGY
NA extraction efficiency was quantified by real time PCR performed in an iCycler iQ5 thermocycler (BioRad, United States) with primers that target the specific regions of the bacteria and phages stated above. A 20 μlreaction mixture contained 5 units of HotTaq DNApolymerase (Sileks, Russia), 1 × PCR buffer (70 mM trisHCl, pH 8.3, 16.6 mM (NH4)2SO4, 2.5 mM MgCl2), 200 μm of each deoxy nucleoside triphosphate (dATP, dCTP, dGTP, dTTP), 2 μl of 1x SYBRGreen I (Invitrogen, United States), and 100 nM of each primer. PCR on biochips. The reaction was performed as described previously [22]. A biochip (Fig. 4a) con tained eight immobilized probes allowing detection of E. coli, B. thuringiensis, Salmonella typhimurium, and phages λ and MS2. To identify the microorganism, we used primers directed against speciesspecific poly morphism of the 16S rRNA gene. In addition, for an increase in specificity, we designed primers specific to the 23S rRNA gene of E. coli, B. thuringiensis, and the fimbrial subunit fimA of fimA S. typhimurium as well. Primer sequences for identification of λ and MS2 phages were complementary to the sequences of gene portions of terminase and coat protein CP, respec
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tively. A biochip also included six gel elements without immobilized probes (negative control “NC”, Fig. 4a) used to assess a background fluorescent signal Iref. Fol lowing amplification, a signal was considered positive if it was above the Iref level at least twofold. RESULTS AND DISCUSSION The development of a unified protocol for automated extraction and purification of NA from biological sam ples. When developed, the protocol for automated extraction and purification of NA had to meet the fol lowing requirements: (1) All steps of sample processing and NA extrac tion should be performed in an automated mode inside a microfluidic module. (2) DNA and/or RNA should be extracted from biological samples of different types including bacte rial cells and viral particles. (3) DNA and RNA should be extracted with a high yield from sample with 104 or more cells and/or viral particles. (4) Microfluidic modules that contain reagents for sample processing should have a long storage period (a year or longer). The first step in biological sample processing is dis ruption (lysis) of bacterial cell walls and viral enve lopes to release NA. Enzymatic lysis involves enzymes such as lysozyme, subtilisin, and proteinase K. Chem ical lysis employs solubilizing and destabilizing agents: sur faceactive compounds (SDS/Triton X100, sodium N lauryl sarcosinate) and chaotropic agents such as guanidine thiocyanate (hydrochloride) or sodium per chlorate at a concentration of 3–6 M [1–3]. Since DNA and RNA must be extracted from bio logical samples that have different structure (bacterial cells and viral particles), we chose a combined two step enzymatic and chemical lysis. The first step of lysis involves sample processing by lysozyme at a con centration of 50 mg/ml that hydrolyzes bacterial cell wall components (peptidoglycans). The second step of lysis occurred in a solution containing proteinase K, a chaotropic agent, and a detergent. Proteinase K hydrolyzes proteins by cleavage of peptide bonds and rapidly inactivates exonucleases, and a chaotropic agent disrupts membranes and capsid envelopes mainly through breaking hydrogen bonds and decreas ing hydrophobic interaction (solubilization of water insoluble molecules). In addition, the presence of a highly concentrated chaotropic agent is a necessary condition for adsorbing NA on silica gel carriers dur ing the next step of extraction and purification. Conditions for the twostep lysis were optimized in experiments with E. coli and B. thuringiensis cells as well as E. coli cells infected with MS2 or λ bacteriophages. The first step of lysis was run at 37°С, which is the tem
perature optimal for lysozyme activity; in the second step, the temperature was increased up to 60°C to pre vent guanidine hydrochloride from crystallization in the solution. Overall, rapid and efficient disruption of cell membranes and viral envelopes was achieved under the following conditions. (1) The first step is the incubation of a sample in 10 mM trisHCl buffer containing 1 mM EDTA and 50 mg/ml lysozyme, pH 8.0, while stirring at 37°С for 10 min. (2) The second step is the incubation of a sample in 10 mM trisHCl buffer containing 1 mM EDTA, 4.5 М guanidine hydrochloride, 1 mg/ml proteinase K, 0.5% sodium Nlauryl sarcosinate, pH 6.5, while stirring at 60°C for 10 min. To prepare a highly purified NA sample, we selected solidphase extraction of NA. It is preferen tial since, besides its simplicity and quickness, this method readily adapts to the format of a microfluidic system. For adsorption, we tried glass microfiber filters GF and silica gel G60 (data not shown). The experi ment involved the use of lysates of bacterial cells and phage particles produced after the procedure of the twostep lysis described earlier. High efficiency of extraction of both DNA and RNA using solidphase carriers in the presence of eth anol was achieved in a sorption buffer. When no etha nol was present, only DNA but not RNA was adsorbed and subsequently eluted. An ethanol concentration of 30 vol % proved to be optimal for efficient extraction and purification of DNA and RNA from lysates using the chosen carriers. Of all the sorbents studied, the most suitable turned out to be silica gel G60 that allows quantitative adsorption and desorption of 103 or more genome equivalents of DNA and RNA. To remove molecules nonspecifically bound to the sorbent, a microcolumn and silica gel with NA adsorbed were successively washed with a mixture of a buffer containing 0.15 M trisНC1, 0.05 M EDTA, 3 M guanidine hydrochlo ride, pH 8.0, and ethanol (up to 50%) and then with 80% ethanol. This washing retains nucleic acids insol uble in ethanol on the sorbent. Nucleic acids were eluted in lowsalt buffer or water. The procedure of selective NA extraction in the microcolumn with the silica gel took less than 10 min. Overall, for extraction and purification of NA of bacterial cells and/or phage (viral particles), we devel oped a unified technique involving a twostep lysis fol lowed by solidphase adsorptiondesorption in the microcolumn with silica gel. Storage of reagents for extraction and purification of NA inside the microfluidic module. Reagent storage within devices is a common problem of all manufac turers of microfluidic systems in which chemical and biochemical reactions and dissolving of reagents and
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reaction mixtures occur [23, 24]. This problem was solved in the following way. Solutions required for sample processing and loaded into module reservoirs contain lysis enzymes (lysozyme and proteinase K) and buffers for lysis and washing with a highly concen trated chaotropic agent (guanidine hydrochloride at a concentration of 3 M or greater). To prevent loss of enzymatic activity, readytouse modules with reagents must be stored at a temperature lower than 10°C; however, this causes the chaotropic agent to crystallize. To avoid enzyme inactivation and salt crys tallization, we suggested that lysis and wash buffer reagents should be stored in a freezedried state. Over all, some microfluidic module reservoirs contain freezedried reagents, others contain their solvents, waterethanol solutions, that could also be stored for a long time. Dissolution of freezedried components occurs during extraction and purification procedure. Freezedried components of the first lysis buffer with lysozyme are dissolved upon the addition of a liquid biological sample into a corresponding reservoir, and freezedried components of the second lysis buffer with guanidine hydrochloride, proteinase K, and sodium Nlauryl sarcosinate are dissolved in the mix ture after the first lysis step. Buffers in a freezedried state inside a microfluidic module could be stored at +4 to +8°С for at least 6 months without a decrease in enzymatic activity of any reagent. The disposable microfluidic module for extraction and purification of nucleic acids from biological sam ples. A circuit schematic and a photo of the module with all reservoirs incorporating all necessary compo nents for lysis of cells, bacteria, and viral particles; for extraction and purification of nucleic acids; and microchannels and valves are shown in Fig. 1. The microfluidic module consists of several con structional elements (Fig. 2). Reservoirs for storage of reaction mixtures, solvents, and sample processing and a microcolumn and microchannels are integrated inside the main working platform. The module closes with the upper and lower lids (panels) equipped with elastic spacers. The upper panel includes 16 openings for solenoid rods, an opening for the sample inlet chamber, seven openings for fixing screws (Fig. 2). The module dimensions (length × width × height) are 94 × 64 × 40 mm. Reaction mixtures and reagents in module reser voirs were moved by delivering pressure into corre sponding reservoirs by a compressor through a network of microchannels and valves (valves a–k in Fig. 1b) formed using module elastic spacers. Required excess pressure was 1.5–2.0 atm. Opening and closing of the valves, which regulate the flow of reaction mixtures and reagents through microchannels and module reser voirs, delivery of pressure into module reservoirs, and APPLIED BIOCHEMISTRY AND MICROBIOLOGY
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heating and mixing of reaction mixtures were executed with devices incorporated in the control unit. The module is designed to process a biological sample of 100–500 μl. The extraction and purification of NA inside the microfluidic module consisted of the following steps: The first lysis step. A biological sample is loaded into inlet chamber 1 with a pipette or another dis penser. Upon opening of the valve a (with the other valves closed) the sample is loaded into reservoir 2 containing freezedried components of the first lysis buffer. In reservoir 2, dissolving of the reaction mix components of the first lysis buffer and the first lysis step occurred simultaneously with the biological sam ple itself being the solvent for the freezedried buffer mixture. The first lysis step was carried out under intense stirring and heating up to 37°C for 10 min. The second lysis step. Through the open valve b (with the other valves closed), after the first lysis step, the reaction mix was driven to reservoir 3 containing the freezedried mix for the second lysis buffer. Dis solving of the freezedried mix for the second lysis buffer and the second lysis step occurred simulta neously with the resulting solution after the first lysis step being the solvent for the freezedried mixture. The second lysis step was also carried out under intense stirring and heating up to 60°C for 10 min. The preparation of the washing buffer. Simulta neously with the second lysis step, the waterethanol solution was moved from reservoir 6 into reservoir 5, which contained the freezedried buffer mixture for washing the solidphase sorbent (valve e is opened; valve f is closed). The freezedried buffer mixture was dissolved under intense stirring. The creation of optimal conditions for binding nucleic acids with a solidphase carrier. After the sec ond lysis step, ethanol from reservoir 4 was added to reservoir 3 with the lysate of the biological sample to reach 30 vol % ethanol concentration (valve c is opened, the other valves are closed). The sorption of nucleic acids on a solidphase sor bent. The reaction mix was directed from reservoir 3 to microcolumn 8 with G60 silica gel. Nucleic acids were adsorbed on the silica gel, and unbound substances were discarded into waste reser voir 11 (valves d and k are opened; the other valves are closed). The washing of a solidphase sorbent. The wash buffer was directed from reservoir 5 to microcolumn 8 and then 80% ethanol was added from reservoir 7. Solutions after washing were driven into waste reser voir 11 (valves f, k and g, k opened, respectively). The elution of nucleic acids off the microcolumn. Nucleic acids were eluted off the solidphase sorbent by driving the elution solution (water or lowsalt ТЕ
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buffer) from reservoir 9 (valves i, j are opened; the other valves are closed). The extracted sample was moved into reservoir 10 for product collection. The outlet port of the module is compatible with a standard 0.2ml microtube that could be used for PCR analysis. The extraction and purification procedure starting from sample loading took less than 40 min. The device for automated extraction and purifica tion of NA. The device for automated extraction and purification of NA includes a microfluidic module and a control unit (Fig. 3). The microfluidic module con tains only reservoirs with reagents and valves for directing gasliquid flows, whereas all necessary con trolling devices that deliver pressure, flow reaction mixtures and reagents in module reservoirs, and heat and stir are located in the control unit. To stir solutions in module reservoirs where necessary, there are niches for stir bars on the bottom (tefloncoated steel sticks). The module prepared for work is installed in the control unit. A biological sample is loaded into the module inlet chamber and then all steps of lysis of cells, microorganisms, and viral particles, purification and elution of NA, are performed sequentially in module reservoirs isolated from the outside environ ment. The sample processing by virtue of this device is computer operated with appropriate software. A disposable microfluidic module is installed into the control unit along the upper and lower positioning
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Fig. 6. Results of realtime PCR on DNA of phage λ as a template extracted from variable dilutions of phage infected E. coli cultures (108–102 per 1 ml of original sam ple) using the standard extraction method (solid line) and microfluidic modules (dotted line). Curves are generated using varied dilutions of cells: 1—108; 2—107; 3—106; 4—105; 5—104; 6—103; 7—102 cell/ml; 8—no template (control).
rails (see Fig. 3) that allow its accurate positioning. Directing of gas and liquid flows in the module is con trolled by 16 valves with electrical control. After instal lation of the module into the control unit and closing the locking mechanism, the module occupies a posi tion in which rods of all 16 solenoids are precisely above the closing module valve heads. A diaphragm pump was used as a compressor capa ble of delivering excess air pressure (1.5–2.0 atm). Heating of module reservoirs in which lysis and solu tion of freezedried reaction mixtures of buffers takes place is accomplished by Peltier elements located under the installation slot of a disposable module in the control unit; it is also the place for magnet devices for stirring solution and reaction mixtures. For general control of Peltier elements, valves, stirring devices, etc. we developed a single microprocessor circuitry. Automated extraction and purification of nucleic acids using a microfluidic module. The developed device based on microfluidic modules allows efficient automated extraction of both DNA and RNA from bacteria and/or viral (phage) particles (Fig. 5). Quan titative evaluation of automated extraction of NA using dilutions of cell cultures in comparison to con ventional assays was carried out by realtime PCR. Results of amplification of the phage λ gene fragment using template DNA of phage λ that was extracted from variable dilutions of phageinfected E. coli cells
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METHOD FOR AUTOMATED EXTRACTION AND PURIFICATION OF NUCLEIC ACIDS Results of automated extraction of E. coli DNA (108 CFU/ml, original sample volume is 0.1 ml) in microfluidic modules (a total of 20 modules) Parameter
DNA Elution buff Concentra D yield, µg er volume, µl tion, ng/µl 260/280
Maximum value
5.5
97
77
1.85
Minimum value
4.9
81
65
1.78
Mean value
5.2
86
69
1.82
Standard deviation
0.4
5
4
0.01
(108–102 per 1 ml of original sample) are given in Fig. 6. Concentration levels of DNA extracted automatically and manually from cultures containing 104–108 CFU/ml dif fered by no more than 20%. Reproducibility of the developed technique was evaluated by adding 0.1 ml E. coli cultures (108 CFU/ml) into the module inlet chamber fol lowed by automated extraction of NA. DNA was quantified spectrophotometrically. Extraction was sequentially performed in 20 modules from a single sample of overnight E. coli culture. According to the results presented in the table, the difference between the yield and the level of DNA extracted in the microfluidic module was less than 15%. Bacterial and phage nucleic acids extracted using the developed technique were used as templates in multiplex PCR on a special biochip (Figs. 4b–4e). A positive amplification signal in a biochip element due to the formation of specific hybridized duplexes between a newly formed PCR product flanked with a free primer from the solution and a product obtained by extending an immobilized probe was detected by the fluorescence of SYBR Green I intercalated in dou blestranded DNA. Fluorescence intensity in ele ments with immobilized probes nonspecific to DNA fragment of interest did not exceed an average back ground value of Iref by more than 20% in all experi ments. At the same time in elements with specific and, consequently, extended primers, signals significantly exceeded Iref values at lest twofold in all experiments, which allowed reliable identification of all microor ganisms and phages tested. The designed device based on a microfluidic mod ule allows rapid (within 40 min) extraction of nucleic acids from cells of microorganisms and/or viruses in an automated mode with minimal loss. Extraction and purification procedure is carried out inside a cartridge separated from the outside environment, which mini APPLIED BIOCHEMISTRY AND MICROBIOLOGY
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mizes the risk of personnel infection. Obtained NA samples could be used for amplification or hybridiza tion on biochips for direct identification of a pathogen in a sample or further molecular genetic analysis. Coupling of automated NA extraction and amplifi cation on hydrogel biochips with realtime detection in a microfluidic module will promote the develop ment of a labonachip diagnostic complex which combines all steps of sample processing and multi parametric genetic analysis to identify, quantify, deter mine drug resistance, and estimate virulent properties of various biological objects. This work is supported by the State Contract with the Ministry of Education and Science of the Russian Federation, no. 02.522.11.2019, and a Program of the US Department of Energy (IPP Grant Assistance Pro gram), project no. RUS21103 6MO04. REFERENCES 1. Protocol Online: DNA Extraction and Purification, http://www.protocolonline.org/prot/Molecular_Biology/ DNA/DNA_Extraction_Purification/index.html. 2. Sambrook, J. and Russel, D.W., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Lab. Press, 2001, vol. 1. 3. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K., Short Pro tocols in Molecular Biology, 5th ed., Chichester: Wiley, 2002. 4. Tan, S.C. and Yiap, B.C., J. Biomed. Biotechnol., 2009, 574398. 5. Wolfe, K.A., Breadmore, M.C., Ferrance, J.P., Power, M.E., Conroy, J.F., Norris, P.M., and Landers, J.P., Electro phoresis, 2002, vol. 23, pp. 727–733. 6. Wu, Q., Bienvenue, J.M., Hassan, B.J., Kwok, Y.C., Giordano, B.C., Norris, P.M., Landers, J.P., and Fer rance, J.P., Anal. Chem., 2006, vol. 78, pp. 5704–5710. 7. Bencina, M., Podgornik, A., and Strancar, A., J. Sep. Sci, 2004, vol. 27, pp. 801–810. 8. Wen, J., Legendre, L.A., Bienvenue, J.M., and Landers, J.P., Anal. Chem., 2008, vol. 80, pp. 6472– 6479. 9. Berensmeier, S., Appl. Microbiol. Biotechnol., 2006, vol. 73, pp. 495–504. 10. Siddiqui, H., Nederbragt, A.J., and Jakobsen, K.S., Clin. Biochem., 2009, vol. 42, pp. 1128–1135. 11. Gijs, M.A., Lacharme, F., and Lehmann, U., Chem. Rev., 2010, vol. 110, pp. 1518–1563. 12. Huska, D., Hubalek, J., Adam, V., Vajtr, D., Horna, A., Trnkova, L., Havel, L., and Kizek, R., Talanta, 2009, vol. 79, pp. 402–411. 13. Whitesides, G.M., Nature, 2006, vol. 442, pp. 368– 373. 14. Haeberle, S. and Zengerle, R., Lab. Chip, 2007, vol. 7, pp. 1094–1110. 15. Mark, D., Haeberle, S., Roth, G., von Stetten, F., and Zengerle, R., Chem. Soc. Rev., 2010, vol. 39, pp. 1153– 1182.
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