Patent Publication Number: US-2019185920-A1

Title: Methods and systems that detect nucleic-acid targets

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims the benefit of Provisional Application No. 62/539,984, filed Aug. 1, 2017. 
    
    
     TECHNICAL FIELD 
     The current document relates generally to the field of nucleic-acid detection and, in particular, to a highly sensitive and specific nucleic-acid-detection method that includes hybridization of a specific nucleic-acid target to a recognition probe, subsequent specific cleavage of the double-stranded target-probe helix at a specific restriction site, and exponential amplification of the enzymatic cleavage accompanied by release of a molecular marker. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows the recognition state of the currently disclosed method. 
         FIG. 2  shows a portion of the amplification stage of the currently disclosed method. 
         FIG. 3  shows another portion of the amplification stage of the currently disclosed method. 
         FIG. 4  shows the amplification chamber. 
         FIG. 5  shows the various alternative amplification probe designs. 
         FIG. 6  shows an alternative amplification probe design. 
         FIG. 7  illustrates one implementation for a DNA-detection 
         FIG. 8  shows a table, Table 1, that provides a list of oligonucleotides used in experimental verification of methods and systems discussed with reference to  FIGS. 1-6 . 
         FIG. 9  shows a schematic of the process of amplification probe attachment to magnetic beads for the ARV(7SP)-pG probe. 
         FIG. 10  shows the dependence of the HRP-generated signal on the incubation time for the two negative control samples. 
         FIG. 11  shows the results of amplification target addition to the beads with immobilized probes after a 15-minute incubation time. 
         FIG. 12  shows the results of amplification target (“SP”) addition to the barrier-separated beads. 
     
    
    
     DETAIL DESCRIPTION 
     Currently Disclosed Methods and Systems for Nucleic-Acid Detection 
     The current document describes a highly sensitive and specific nucleic-acid detection method which includes: (1) hybridization of a specific nucleic-acid target to a recognition probe, an oligonucleotide immobilized on solid support; (2) subsequent specific cleavage of the double-stranded target-probe helix at a specific restriction site by a corresponding restriction endonuclease (“REase”); and (3) exponential amplification of the enzymatic cleavage accompanied by release of a molecular marker into a reaction solution. Nucleic acids include naturally occurring deoxyribonucleic acids and ribonucleic acids, but may also include various types of non-naturally-occurring nucleic acids, such as nucleic acids containing non-naturally-occurring pyrimidine and purine bases, sugars, and backbone moieties. 
     In certain implementations, the specific recognition probe is single-stranded and is designed to have a recognition part, or a segment of a first type, that is complementary to the target, and an amplification part, or amplification segment, that consists of two or more identical subsequences of a second subsequence type. In certain implementations, the recognition probe is bound to a solid support in a recognition-reaction chamber. The solid support may be nanoparticles, beads, a substrate, or immobile-phase components of a gel. The first-type segment, complementary to the target, is proximal to a first end of the recognition probe adjacent to the solid support and the two or more identical subsequences of the second subsequence type are adjacent to a second end of the recognition probe. The recognition probe can be denoted: “ss-S 1 -S 2  . . . S 2  where S 1 |target.” In this symbolic representation of the recognition probe, “ss” indicates the solid support, “S 1 ” indicates the segment of the first type, “S 2 ” indicates a segment of the second type, “ . . . ” represents 0, 1, or more segments of the same type as the segments adjacent to “ . . . ” in the symbolic representation, and “S 1 |target” indicates that the segment of the first type, S 1 , is complementary to the target. 
     In certain implementations, an amplification reaction chamber contains two different types of solid-support-bound single-stranded oligonucleotide amplification probes and corresponding REases, where, again, the solid support may be nanoparticles, beads, or a substrate, or immobile-phase components of a gel. In alternative implementations, there may be additional or fewer chambers. 
     In certain implementations, a first type of amplification probe includes a third type of subsequence proximal to a first end of the first-type amplification probe adjacent to the solid support and at least two subsequences of a fourth type proximal to a second end of the first-type amplification probe. The first type of amplification probe can be denoted, using the same representation conventions described above in the representation of the recognition probe: “ss-S 3 -S 4  . . . S 4  where S 3 |S 2 .” 
     In certain implementations, a second type of amplification probe includes a fifth type of subsequence proximal to a first end of the second-type amplification probe adjacent to the solid support and at least two subsequences of the second type proximal to a second end of the second-type amplification probe. The second type of amplification probe can be denoted, using the same representation conventions described above in the representation of the recognition probe: “ss-S 5 -S 2  . . . S 2  where S 5 |S 4 .” 
     The subsequence of the second type of the second type of amplification probe is complementary to the fourth type of subsequence of the first type of amplification probe and the third type of subsequence of the first type of amplification probe is complementary to the second type of subsequence of the recognition probe. The third type of subsequence of the first type of amplification probe and the fifth type of subsequence of the second type of amplification probe contain restriction sites specific for enzymatic cleavage by first and second types of amplification REases, respectively. Both types of amplification probes have a molecular marker attached to their second ends. The first type of subsequence of the recognition probe contains a restriction site specific for enzymatic cleavage by a recognition REase. 
     Upon recognition-REase cleavage of the recognition probe, the released second-type segments are transported into the amplification chamber, in certain implementations. There, a second-type segment released by recognition-REase cleavage of the recognition probe hybridizes with the third type of subsequence of the first type of amplification probe and the resultant double-stranded hybrid is cleaved the first type of amplification REase. This enzymatic cleavage of the first type of amplification probe leads to the release of multiple copies of the fourth type of subsequence of the first type of amplification probe and the molecular marker into the reaction solution. The released fourth-type segments then hybridize with fifth-type of subsequences of the second type of amplification probe. The hybrids are then cleaved by the second type of amplification REase. Each cleavage of the second type of amplification probe results in the release of multiple copies of the second type of sub-segment of the second type of amplification probe, also initially present in the recognition probe, and the molecular marker. In turn, each released second-type subsequence of the second type of amplification probe hybridizes with the third type of subsequence of the first type of amplification probe causing cleavage of the first type of amplification probe and release of multiple fourth-type subsequences of the first type of amplification probe and the molecular marker. Thus, enzymatic cleavage of the two types of amplification probes accelerates exponentially, resulting in an exponential increase in the concentration of free molecular marker in the reaction solution. 
     The currently disclosed method consists of two stages: (1) a recognition stage; and (2) an amplification stage.  FIG. 1  shows the recognition stage of the currently disclosed method. A recognition probe  102  is immobilized on a solid surface  104 . The recognition probe  102  is an oligonucleotide molecule that includes a recognition subsequence  106 , or first type of subsequence, complementary to all or a portion of the target  116  and that contains a restriction site  108  specific for the recognition REase  110 . The recognition subsequence  106  is attached to the solid support via either its 3′ or 5′ end. In other words, the recognition probe may have one of two possible orientations corresponding to 5′-to-3′ and 3′-to-5′, where 3′ and 5′ refer to hydroxy groups of the ribose subunits of the nucleotide monomers within the oligonucleotides. The recognition probe  102  additionally contains a subsequence  112  containing one or more copies of a second subsequence  114 . The recognition stage involves hybridization of the target molecule  116  to subsequence  106  of the probe  102 . The target molecule  116  is a single-stranded nucleic-acid molecule that carries at least one subsequence complementary to subsequence  106  of the recognition probe  102 . Upon hybridization of the target  116  to the recognition probe  102 , enzymatic cleavage of the target/restriction-probe duplex at the restriction site  108  by the recognition REase  110  occurs. This cleavage releases the one or more copies of the second subsequence  114  from the solid support into the reaction solution. Thus, following cleavage, the reaction solution contains oligonucleotide molecules  112  that each includes one or more copies of the second subsequence  114 . The amount of released oligonucleotide molecules  112  is proportional to the amount of target molecules to which the recognition probes is exposed. 
       FIG. 2  shows a portion of the amplification stage of the currently disclosed method. The released oligonucleotide molecules  112  can either passively migrate or can be mechanically transported from a first reaction chamber, or recognition environment, to a second chamber, or amplification environment. In certain implementations, connected reaction chambers are employed, while in alternative implementations, probes that may deleteriously interact are spatially separated from one another in local environments, such as well-defined areas of a substrate or the surfaces of different beads or other different surfaces. The released oligonucleotide molecules  112  encounter a first type of amplification probe  202  that is bound, via a first end, to a solid support  201 . A molecular marker  204  is attached to a second end of the first type of amplification probe  202  via a linker  206 . The first type of amplification probe  202  is an oligonucleotide molecule that includes a third subsequence  208  complementary to the second subsequence  114  of the oligonucleotide molecules  112  released in the recognition stage. The third subsequence  208  contains a restriction site  210  specific for a first type of amplification REase  212 . The first type of amplification probe  202  further includes a subsequence  214  that includes one or more copies of a fourth subsequence  216 . Upon hybridization of an oligonucleotide molecule  112 , through a component subsequence  114 , to the first type of amplification probe  202 , enzymatic cleavage of the released-oligonucleotide-molecule/first-type-amplification-probe duplex at the restriction site  210  by the first type of amplification REase  212  occurs. This cleavage leads to the release of subsequence  214  of the first type of amplification probe  202  as well as the attached molecular marker  204  from the solid support into the reaction solution. Thus, following enzymatic cleavage of the released-oligonucleotide-molecule/first-type-amplification-probe duplex, the reaction solution contains oligonucleotide molecules  214  that include the fourth type of subsequence  216 . When the oligonucleotide molecules  214  include more than one copy of subsequence  216 , the oligonucleotide molecules  214  again hybridize with another first-type amplification probe  202  via another subsequence  216 . This hybridization again leads to an additional cleavage of a first-type amplification probe  202 . Thus, the number of released oligonucleotide molecules  214  is proportional to the number of copies of subsequence  216  in the amplification chamber. 
       FIG. 3  shows another portion of the amplification stage of the currently disclosed method. The amplification chamber, or amplification environment, further contains a solid support  302  with an immobilized second type of amplification probe  304 . The molecular marker  204  is attached to one end of the second-type amplification probe  304  via linker  206 . The second-type amplification probe  304  is an oligonucleotide molecule that consists of a fifth type of subsequence which is complementary to the fourth type of subsequence  216 . The fifth type of subsequence includes a restriction site  308  specific for a second type of amplification REase  310 . The second-type amplification probe  304  additionally includes a subsequence  312  that includes one or more second-type subsequences  114 . The oligonucleotide molecules  214  released from the first-type amplification probe passively migrate or are mechanically transported to the second-type amplification probe  304 . Upon hybridization of the oligonucleotide molecules  214  via component subsequences  216  to the fifth type of subsequence  306  of the second-type amplification probe  304 , enzymatic cleavage of the released-oligonucleotide-molecule/second-type-amplification-probe duplex at the restriction site  308  by the second type of amplification restriction REase  310  occurs. This cleavage leads to release of the subsequence  312  and the attached molecular marker  204  from the solid support into the reaction solution. Thus, following enzymatic cleavage of the of the released-oligonucleotide-molecule/second-type-amplification-probe duplex, the reaction solution contains contains oligonucleotide molecules that include one or more copies of the fourth type of subsequence  114 . When the oligonucleotide molecules  214  contain more than one copy of the subsequence  216 , the remaining portion of oligonucleotide molecules  214  again hybridize with another second-type amplification probe  304  via subsequence  216 . This hybridization leads to additional cleavage of the second type of amplification probe  304 . Thus, the number of released oligonucleotide molecules  312  is proportional to the number of oligonucleotide molecules  214  released during the first amplification stage described with reference to  FIG. 2 . The released oligonucleotide molecules  312  passively migrate, or are mechanically transported, back to the first type of amplification probe  202 , hybridize with first type of amplification probe  202  via the second type of subsequence  208 , and the hybridization and cleavage discussed with reference to  FIG. 2  occurs again. During each subsequent cycle, additional molecular marker  204  is also released into the reaction solution together with oligonucleotide molecules  214  and  312 . Upon completion of each cycle of oligonucleotide-molecule  312  release, the number of released oligonucleotide molecules  312  is equal to a×b, where a is the number of oligonucleotide molecules  214  released at the previous cycle and b is the number of fourth-type subsequences  216  in each oligonucleotide molecule  214 . At the same time, upon completion of each cycle, the number of released oligonucleotide molecules  214  is equal to c×d, where c is the number of oligonucleotide molecules  312  released at the previous cycle and d is the number of second-type subsequences  114  in each oligonucleotide molecules  312 . Thus, the reactions involving the oligonucleotide molecules  214  and  312  and the molecular marker  204  result in an exponential increase in the concentration of the molecular marker  204  in the reaction solution. 
       FIG. 4  shows one implementation of an amplification chamber. The amplification chamber is designed to provide separation of the first and second types of amplification probes  202  and  304  from the direct contact with each other. Thus, hybridization of the second-type subsequence  114  of the probe  304  with the third-type subsequence  208  of the first type of amplification probe  202  is impossible without the enzymatic cleavage and release of the oligonucleotide molecules  312 . At the same time hybridization of the fourth-type subsequence  216  of the first type of amplification probe  202  with the fifth-type subsequence  306  of the second type of amplification probe  304  is impossible without the enzymatic cleavage and release of the oligonucleotide molecules  214 . This separation is achieved by either physical separation of the solid supports  201  and  302  or localization of the first and second types of amplification probes  202  and  304  on a common solid support that does not permit physical contact between the two types of amplification probes. 
     After the enzymatic release of oligonucleotide molecule  312  by amplification REase  310  and oligonucleotide molecule  214  by amplification REase  212 , the oligonucleotide molecules migrate to amplification probes  202  and  304 , respectively. This migration results in hybridization of the second-type subsequence  114  with third-type subsequence  208  and hybridization of the fourth-type subsequence  216  with the fifth-type subsequence  306  followed by subsequent enzymatic cleavage of amplification probes  202  and  304 . Since the oligonucleotide molecules  312  include two or more copies of the second-type subsequences  114 , two or more enzymatic cleavages of the amplification probes  202  occur. Thus, each initial cleavage of the second-type amplification probe  304  is followed by two or more cleavages of the first-type amplification probe  202 . This provides for a two-fold amplification of the signal per cycle. The initial cleavage of the first-type amplification probe  202  and the release of oligonucleotide molecules  214  provides for the signal amplification in a similar way. Since the oligonucleotide molecules  214  includes of two or more fourth-type subsequences  216 , two or more enzymatic cleavages of the second-type amplification probe  304  occur after oligonucleotide molecules  214  migrate to the second-type amplification probe  304 . 
       FIG. 5  shows the various alternative amplification probe designs. The amplification probes  202  and  304  may be designed as linear molecules, as shown in  FIGS. 2 and 3 , but may alternatively be designed as branched molecules in which the multiple copies of the second-type subsequences  114  are linked to the fifth-type subsequence  306 , forming oligonucleotide molecule  312  of the second-type amplification probe  304 , and multiple fourth-type subsequences  216  are linked to third-type subsequence  208 , forming oligonucleotide molecule  214  of the first-type amplification probe  202 . 
       FIG. 6  shows an additional alternative amplification probe design. The amplification probes  202  and  304  include carriers  602  and  604 , respectively, linked to a third-type subsequence  208  and fifth-type subsequence  306 . The carrier surface is modified with multiple fourth-type subsequences  216  and second-type segments  114 , respectively, and with molecular markers  204 . The carriers  602  and  604  can be particles of nano- and micro size, or protein, lipid, synthetic-polymer, and other types of macromolecules. Each carrier can contain a large number of fourth-type subsequences  216  and second-type segments  114 , from hundreds to thousands or more, thus providing for high amplification coefficients. 
     The solid support for probe immobilization can be a solid, such as a metal or plastic, a gel, such as gelatin, alginate, etc., and various types of beads, micro-particles, nano-particles, and mixed-scale particles. An oligonucleotide probe can be linked to the gel via a biotin-streptavidin interaction, amino groups, sulfhydryl groups, aldehyde groups, etc. 
     The molecular marker can be one or more of: (1) a fluorescent label, such as fluorescein isothiocyanate, for fluorescent detection; (2) an enzyme label, such as horseradish peroxidase or alkaline phosphatase, to provide further enzymatic conversion of an enzymatic substrate for optical or electrochemical detection; and (3) an electrochemical label, such as ferrocene derivatives, to provide a substrate for electrochemical conversion and detection. 
       FIG. 7  illustrates one implementation of an amplification cell. The amplification cell  702  contains carriers  704  and  706  with amplification probes immobilized to their surfaces. The carriers  704  and  706  are separated by a barrier  708 . The barrier  708  does not permit carriers  704  and  706  to interact with each other. At the same time the barrier  708  is permeable for all other participants of the amplification process: enzymes, oligonucleotides, ions, etc. The carriers are kept in suspended conditions during the amplification reaction by either alternating a magnetic field, by ultrasonic treatment, or by mechanical agitation. 
     Experimental Results 
     Oligonucleotide Probes for Recognition and Amplification 
       FIG. 8  shows a table, Table 1, that provides a list of oligonucleotides used in experimental verification of methods and systems discussed with reference to  FIGS. 1-6 . Table 1 provides a list of applied oligonucleotides purchased from Integrated DNA Technologies (Skokie, Ill.). The oligonucleotide bi-pC-ST-SP, with a biotinylated 5′ end, was used as a probe for the recognition stage to detect the target AST. The oligonucleotides ARV-7SP-pG and ASP-7RV-pG were used as templates to generate biotinylated amplification probes using the polymerase chain reaction (“PCR”). Biotinylated oligonucleotides bi-pA-ARV(GT) and bi-pA-ASP(CTAT), and the biotin-free pC, were used as PCR primers. The thiol pC oligonucleotide was used to obtain an HRP linked tag for attachment of HRP to 3′ ends of amplification probes. 
     HRP Conjugate Preparation 
     To attach the horseradish peroxidase (“HRP”) marker to the amplification probes, HRP was conjugated to the oligonucleotide pC. The conjugation reaction was carried out with an excess of HRP compared to the oligonucleotide, producing a final conjugate concentration equivalent to the initial oligonucleotide pC concentration. 
     Amplification Probes Preparation Via PCR 
     The amplification probes were obtained by PCR using the Phusion® High-Fidelity PCR Kit (New England Biolabs, Ipswich, Mass.). The oligonucleotides ARV-7SP-pG and ASP-7RV-pG were used as templates at 1 nM concentrations. Primer pairs: bi-pA-ARV(GT) plus pC, and bi-pA-ASP(CTAT) plus pC were used for amplification of these templates, respectively. 
     PCR was performed under the following conditions: 10 sec at 95° C. denaturing, 20 sec at 58° C. annealing, and 20 sec at 72° C. extension, for 40 cycles. The PCR product was then purified by using the Invitrogen ChargeSwitch®-Pro PCR Cleaning Kit (Thermo Fisher Scientific Inc., Rockford, Ill.), and applied to a Micro Bio-Spin column with Bio-Gel P-6 (“P-6 column”) (Bio-Rad, Hercules, Calif.) pre-equilibrated with phosphate buffered saline (“PBS”). The product volume was then adjusted to the initial PCR product volume before purification by adding PBS. 
     Note: Both biotinylated primers bi-pA-ARV(GT) and bi-pA-ASP(CTAT), and thus, the resultant amplification probes contain a spacer sequence of 12 adenines to increase the distance between the probe and the attachment surface (bead). 
     Amplification Probe Attachment to Magnetic Beads Surface 
     Hydrophilic Streptavidin Magnetic Beads suspension (400 uL of 4 mg/mL stock, New England Biolabs) was settled and washed 2 times with 400 uL PBS. Then the beads were resuspended in 400 uL of 200 mM sodium hydroxide for 5 min to remove weakly bound streptavidin. The beads were then washed twice with 10×PBS and 3 times with 1×PBS. After drying, the beads were mixed with 400 uL of the purified PCR product (diluted 1:1 with PBS). The bead suspension was then incubated for 5 hours at room temperature in a Labquake Shaker Rotisserie (Thermo Fisher Scientific) set at 8 rpm. After incubation, the beads were washed 5 times with PBS and then treated with 400 uL of 200 mM sodium hydroxide for 5 min to denature double-stranded DNA. The beads were washed twice with 400 uL 10×PBS and 6 times with 1×PBS. Then the bead suspension was mixed with 800 uL PBS containing 150 nM HRP-pC conjugate and 0.5 mg/mL of BSA and incubated at room temperature overnight. Finally, the beads were washed 10 times with 800 uL of PBS and stored at 4° C. until use. The final working concentration of the beads in PBS was 2 mg/mL.  FIG. 9  shows a schematic of the process of amplification probe attachment to magnetic beads for the ARV(7SP)-pG probe. 
     Amplification Test for Mixture of Beads with Two Different Amplification Probes 
     10 uL of beads (2 mg/mL) modified with either ASP-7RV-pG-HRP or ARV-7SP-pG-HRP were placed into separate 0.5 mL Eppendorf tubes. The beads were washed twice with 10 uL of CutSmart buffer (New England Biolabs, Ipswich, Mass.), and the supernatant was removed. 
     Next, 10 uL of CutSmart buffer containing a particular concentration of SP target oligonucleotide was added to the ASP-7RV-pG-HRP beads. The ARV-7SP-pG-HRP beads were used to add 10 uL of CutSmart buffer containing two REases, EcoRV and SspI (0.8 U/uL each). Finally, both bead suspensions were mixed together and incubated at 37° C. in a rotisserie. After incubation, the beads were settled, and the supernatant containing the cleaved HRP was collected for the signal measurement. The supernatant (18 uL) was placed into a microplate well and mixed with 100 uL of BioFX TMB One Component HRP Microwell Substrate (SurModics, Eden Prairie, Minn.) to start the HRP colored reaction. Negative controls (three) were prepared as no-target added samples. The blue color HRP-generated signal forming after 10-12 min incubation at room temperature was measured colorimetrically at the wavelength of 655 nM (OD 655 ) with a Bio-Rad iMark Microplate Reader. 
       FIG. 10  shows the dependence of the HRP-generated signal on the incubation time for the two negative control samples containing no-target added beads supplemented with either single SspI REase ( 1002  in  FIG. 10 ), or both EcoRV and SspI REases ( 1004  in  FIG. 10 ). Since both types of the beads are present in each suspension, the probes on the bead surfaces can hybridize to each other. Thus, the SP part of ARV-7SP-pG-HRP probe can hybridize to the ASP part of ASP-7RV-pG-HRP probe, and vice versa. Such hybridization of the SP and ASP complementary sequences is followed by the enzymatic cleavage of the probes by SspI enzyme. The cleavage releases the probes into the reaction solution, which can then be detected by measuring the HRP signal.  FIG. 2  shows that, for the single REase system ( 1002  in  FIG. 10 ), a slight increase of the HRP signal is observed after about 30 min incubation. For the two REase system ( 1004  in  FIG. 10 ), a sharp increase of the signal is observed in the direct correlation with the incubation time increase. This prominent signal increase in the no-target added negative controls demonstrates that even very low level of random target-independent probe cleavage is triggering a cascade of exponential amplification reactions. A final curve  1006  shows that, when the two sets of beads are separated by a barrier, almost no increase in the HRP signal is observed. 
       FIG. 11  shows the results of amplification-target (“SP”) addition to the beads with immobilized probes after 15 min incubation time. The HRP signal was generated in either a single REase system ( 1102  in  FIG. 11 ) or a two REase (EcoRV and SspI) ( 1104  in  FIG. 11 ) system. The X-axis shows the target concentrations (M), and the Y-axis shows the background-subtracted HRP signal values, with the background calculated as the mean signal generated for triplicate no-target added negative controls. For normalization and comparison of the sample series, the HRP signal values are expressed as the percentages of the maximum background-subtracted OD 655  corresponding to each series. Error bars show standard deviations.  FIG. 11  shows that, for the single-REase system ( 1102  in  FIG. 11 ), the lower detection limit is in the nanomolar concentration range, which is expected, since it is incapable of signal amplification. The two REase system ( 1104  in  FIG. 11 ) has a detection limit in the 10-100 attomolar range due to the exponential signal amplification. 
     Amplification Test for Barrier-Separated Beads with Two Different Amplification Probes 
     To prevent formation of high negative control signals described above with reference to curve  1004  in  FIG. 10 ), the amplification beads with probes ASP-7RV-pG-HRP and ARV-7SP-pG-HRP were separated with a physical barrier of glass filters permeable only to the soluble compounds. A 5-mm-diameter disk made of glass fiber of filter grade A (I.W. Tremont Co., Inc, Hawtrone, N.J.) was placed into a microplate well. Next, 10 uL of ARV-7SP-pG-HRP modified bead suspension (2 mg/mL in CutSmart buffer) was deposited onto the filter. This was overlain with a second filter and topped with 10 uL of ASP-7RV-pG-HRP modified beads (2 mg/mL in CutSmart buffer). A magnetic field was then applied for 10 seconds to the bottom of the microplate to fix the beads on the top of filters. Next, 20 uL of the CutSmart buffer containing the SP target oligonucleotide was added to the well. Finally, 20 uL of CutSmart buffer containing two REases, EcoRV and SspI (0.8 U/uL each) was also added to the well. The resultant assembly microplate was then incubated for various time intervals at 37° C. with shaking at 90 revolutions per min. After incubation, 20 uL of PBS were added to the well and mixed with the reaction solution, and 50 uL of the mix was then collected. The residual beads were settled from the reaction mixture and 35 uL of the supernatant were placed into a microplate well and mixed with 150 uL of BioFX TMB One Component HRP Microwell Substrate to start the HRP colored reaction. The blue color HRP-generated signal that formed after 30-40 mM incubation at room temperature was measured colorimetrically at a wavelength of 655 nM (OD 655 ). 
       FIG. 10  shows the dependence of the HRP-generated signal on the incubation time for the negative control samples. These samples contain no-target added beads supplemented with both EcoRV and SspI REases ( 1006  in  FIG. 10 ). Unlike the bead mixture ( 1002  in  FIG. 10 ), the barrier-separated beads demonstrate almost no release of HRP even for a long incubation time of 90 min. 
       FIG. 12  shows the results of amplification target (“SP”) addition to the barrier-separated beads with immobilized probes after 80 min incubation time ( 1202  in  FIG. 12 ). The HRP signal was generated in a two-REase (EcoRV and SspI) system. The X-axis shows the target concentrations (M) and the Y-axis shows the background-subtracted HRP signal values, with the background calculated as the mean signal generated for three replicates of no-target added negative controls. For normalization and comparison of the sample series, the HRP signal values are expressed as the percentages of the background-subtracted signal obtained at the highest concentration, 10 nM, corresponding to each series. Error bars show standard deviations. The two REase system with the barrier-separated beads ( 1202  in  FIG. 12 ) has a detection limit in the 10 attomolar range due to the exponential signal amplification. However, the barriers separating the beads with different probes add diffusion limitations, requiring longer incubation times compared to the bead mixture-based system discussed with reference to  FIG. 11 . The overall HRP generated signal is also lower for the barrier-based system compared to the mixture-based one. 
     Recognition Probe Attachment to Magnetic Beads 
     A Hydrophilic Streptavidin Magnetic Bead suspension (200 uL of 4 mg/mL stock suspension) obtained from New England Biolabs (Ipswich, Mass.) was settled and washed 2 times with 200 uL PBS. The beads were then resuspended in 200 uL of 200 mM sodium hydroxide for 5 min to remove weakly bound streptavidin. The beads were then washed twice with 10×PBS and 3 times with 1×PBS. After removing the supernatant, the beads were mixed with 200 uL of the 1.6 nM solution of bi-pC-ST-SP oligonucleotide, the recognition probe, in PBS. The bead suspension was then incubated for 5 hours at room temperature in a Labquake Shaker Rotisserie (Thermo Fisher Scientific) at 8 rpm. After incubation, the beads were washed 5 times with PBS and then treated with 200 uL of 200 mM sodium hydroxide for 5 min to remove non-specifically and weakly bound probes. Finally, the beads were washed twice with 200 uL 10×PBS and 6 times with 1×PBS. 
     Combination of Recognition and Amplification Tests 
     The recognition beads were transferred to a saline-sodium-phosphate-EDTA (“SSPE”) buffer to prepare a 2 mg/uL suspension. 10 uL of the bead suspension was then dried in a magnetic stand and supplemented with 40 uL of SSPE buffer containing the AST target. The reaction suspension was incubated for 40 min at ambient temperature in a rotisserie to achieve target-to-probe hybridization. The beads were then washed twice with SSPE buffer and 3 times with CutSmart buffer. Then, 20 uL solution of 0.2 U/uL of StuI REase was added and the beads were resuspended and incubated for 40 min at 37° C. in a rotisserie. After enzymatic cleavage of the pC-ST-SP probe, the supernatant containing the released SP oligonucleotide was collected and used for signal amplification, as described above in the section. The calibration curve obtained for AST target detection using the described procedure is shown in  FIG. 12  ( 1204  in  FIG. 12 ). This plot is similar to the plot generated using the amplification step only, also shown in  FIG. 4  ( 1202  in  FIG. 12 ). For both plots the detection limit is in the attomolar concentration range. 
     Although the invention has been explained in relation to its preferred embodiment, it is to be other possible modifications and variations can be made without departing from the spirit and scope of the invention. For example, as discussed above, many different types of apparatus may be used, various alternative apparatuses including one, two, or more interconnected reaction chambers, with solid-support-immobilized probes and cleavage products moved between chambers by mechanical solution transfer, applied magnetic fields, passive diffusion, and by other techniques. A single-chamber implementation is possible in addition to the barrier-implemented double chamber shown in  FIG. 7 . In additional implementations, a small fluid volume may be moved across a substrate, without the need for an enclosing chamber.