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WO2008119018A1 - Capture et libération sélectives d'analytes - Google Patents

Capture et libération sélectives d'analytes Download PDF

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Publication number
WO2008119018A1
WO2008119018A1 PCT/US2008/058433 US2008058433W WO2008119018A1 WO 2008119018 A1 WO2008119018 A1 WO 2008119018A1 US 2008058433 W US2008058433 W US 2008058433W WO 2008119018 A1 WO2008119018 A1 WO 2008119018A1
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Prior art keywords
analyte
aptamer
released
release
temperature
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WO2008119018A8 (fr
Inventor
Qiao Lin
Jingyue Ju
Milan N. Stojanovic
Thaihuu Nguyen
Chunmei Qiu
Renjun Pei
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Columbia University in the City of New York
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Columbia University in the City of New York
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Publication of WO2008119018A1 publication Critical patent/WO2008119018A1/fr
Publication of WO2008119018A8 publication Critical patent/WO2008119018A8/fr
Anticipated expiration legal-status Critical
Priority to US12/568,651 priority Critical patent/US20100151465A1/en
Priority to US13/652,214 priority patent/US9250169B2/en
Priority to US14/978,716 priority patent/US20160169780A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the present application relates to, but is not limited to, selective capture and release of analytes.
  • the present application relates to minimally invasive extraction, purification and concentration (PC) of analytes.
  • sample preparation One component in these devices is sample preparation, which involves extraction and PC of applicable analytes.
  • sample PC Some techniques for sample PC have employed solid-phase (SP) gels for retention of target molecules.
  • SP devices solid-phase
  • hydrophobic and ion-exchange SP device are limited because they extract impure compounds with similar physical or chemical properties as the target.
  • introducing impurities can be problematic.
  • elution of molecules using harsh pH or solvent gradients is common in SP devices. For certain biomedical applications, these elution schemes can present potential health hazards.
  • Some embodiments include components for capture and selective release of an analyte.
  • components including a solid phase, an aptamer functionalized on the solid phase for binding the analyte, and a temperature regulator for setting a temperature to a set point, such that the analyte is released from the aptamer at the set point.
  • the analyte can initially exist in an impure form and the impurities can be removed with a washing solution after the analyte is bound to the aptamer.
  • the analyte can include an oligonucleotide.
  • the solid phase can include a microbead.
  • the analyte can be in an aqueous solution.
  • the components can further include a collector for collecting the released analyte; and a detector for measuring the amount of analyte released.
  • the collector can include a spotting well.
  • the detector can be a mass spectrometer.
  • the components can further include a microchannel for receiving the released analyte and directing the released analyte through a hydrophobic valve.
  • the components can be incorporated on a microfluidic chip platform.
  • the methods include binding the analyte to an aptamer, the aptamer functionalized on a solid phase, and setting the temperature of the aptamer such that the analyte is released from the aptamer.
  • the procedure can further include introducing the analyte to the aptamer in an impure form and washing the bound aptamer analyte complex to remove impurities.
  • the procedural elements can be repeated so that the amount of bound analyte is increased.
  • the procedure can further include collecting and detecting the analyte.
  • the detecting can include performing mass spectrometry on the released analyte or detecting fluorescence intensity.
  • the methods include functionalizing a solid phase with an aptamer, introducing the analyte to the aptamer in an impure form, binding the analyte to the aptamer, and washing the bound aptamer analyte complex to remove impurities.
  • the procedural elements can be repeated so until a desired analyte concentration is reached, and the temperature of the aptamer can be set such that the analyte is released from the aptamer.
  • Fig. 1 is a schematic drawing showing an example device according to one embodiment of the described subject matter.
  • Fig. 2 depicts an example according to an embodiment of the described subject matter.
  • the figure shows a fabrication technique: 2a-c show polydimethysiloxane (PDMS) channel techniques and 2d shows a package.
  • PDMS polydimethysiloxane
  • Fig. 3 depicts an example according to an embodiment of the described subject matter. The figure shows the relationship of fluorescence signal to TO-AMP concentration in solution.
  • Figs. 4(a)-(b) depict exemplary devices according to the described subject matter, with Fig. 4(a) showing device 1: 200 nM injections, and Fig. 4(b) showing device 2: 500 nM injections.
  • Fig. 5 depicts thermal release of AMP from bead surfaces incubated with 10 ⁇ M injection according to an embodiment of the described subject matter
  • Figs. 6 depict extraction of AMP after release at (a) 75 0 C, (b) 85°C, and (c) 95°C according to exemplary embodiments of the described subject matter
  • Figs. 7(a)-(b) depict (a) chemical structure of bio-ATP-40-1 aptamer and (b) molecular structure of TO-AMP according to exemplary embodiments of the described subj ect matter
  • Fig. 8 depicts an example layout of the microfluidic SPE device used for this demonstration according to an exemplary embodiment of the described subject matter
  • Figs. 9(a)-(f) depict a simplified device flow: (a-d) a microchannel realized with standard soft lithography techniques and (e & f) a package according to exemplary embodiments of the described subject matter.
  • Line A-A' in the schematic of Fig. 8 is a cross-section reference
  • Fig. 10 depicts a bright-field micrograph of the chamber, including a magnified area for fluorescence imaging and processing according to an exemplary embodiment of the described subject matter
  • Figs. l l(a)-(b) depict time resolved extraction of TO-AMP (400 nM) by ATP-aptamer according to an exemplary embodiment of the described subject matter. Measurements are made in the (A-A') direction of the linked micrograph as shown in Fig. 11 (a). At each time level, fluorescence intensity is sampled, averaged, and normalized to produce a single value point for each interval.
  • Figs. 12(a)-(e) depict micrographs displaying SPE extraction of 3 concentrations of TO-AMP according to exemplary embodiments of the described subject matter: (a) baseline fluorescence; (b) 400 nM; (c) 500 nM; and (d) 10 ⁇ M.
  • Figs. 13(a)-(b) depict controlled release of TO-ATP and regeneration of an exemplary SPE device of the described subject matter (baseline colinear w/horizontal axis).
  • Fig. 13(a) shows competitive displacement with ATP (800 ⁇ M & 3.2mM).
  • Fig. 13(b) presents thermally induced release and regeneration. Single- valued points are obtained similarly to time-resolved data.
  • Figs. 14(a)-(b) depict, schematics of an example device according to an embodiment of the described subject matter.
  • Fig. 14(a) is a top view.
  • Fig. 14(b) is an I-I cross-sectional view (dashed line refers to a hidden waste outlet.).
  • the Chip dimensions are 2x2x0.16 cm (lxwxh).
  • Figs. 15(a)-(e) depict an example according to an embodiment of the described subject matter.
  • Figs. 16(a)-(b) depict operation of a passive valve: (a) fluorescein solution flowing through the waste outlet bypassing valve and (b) valving fluorescein solution through the valve according to an exemplary embodiment of the described subject matter.
  • Fig. 17 depicts spot size on a MALDI plate as a function of flow rate used to transfer the sample to the deposition well according to an exemplary embodiment of the described subject matter.
  • Figs. 18(a)-(b) depict MS from (a) a 0.1 ⁇ M injected sample and (b) a 1.0 ⁇ M injected sample according to exemplary embodiments of the described subject matter.
  • Fig. 19 depicts MS from an injected sample of AMP, CTP, UTP, & GTP (1 ⁇ M each) where AMP is isolated according to an exemplary embodiment of the described subject matter.
  • Figs. 20(a)-(b) depicts MS from a sample spot obtained from (a) 20 injections; (b) 200 injections of 10 nM AMP solution according to exemplary embodiments of the described subject matter.
  • the presently described subject matter will now be described in detail with reference to the Figures in connection with the illustrative embodiments.
  • the described subject matter includes techniques and components for minimally invasive, selective capture and release of analytes.
  • An aptamer is selected for its binding affinity with a particular analyte(s).
  • the aptamer is functionalized on a solid phase, for example, microbeads, polymer monolith, microfabricated supports, etc.
  • the analyte is allowed to bind to the aptamer, for example, in a microchamber.
  • a temperature control sets the temperature to an appropriate temperature at which the captured analyte is released.
  • Affinity binding includes the reaction between a ligand and a specific receptor, such as an antigen and antibody or enzyme and substrate.
  • the strong specificity stems from the ligand and receptor being ideally suited to one another both electrostatically and spatially. Additionally, ligand and receptor binding can be reversed by such stimuli as heat and ionic strength.
  • antibody/lectin devices are one affinity pair
  • high-affinity aptamers derived from nucleic acid are drawing increased attention because they can be synthesized selectively towards any target molecule. They offer long-term stability, relatively straightforward synthesis, and the capability of modifiable end-chains to facilitate labeling or immobilization.
  • aptamers can reversibly bind to their targets within an aqueous environment, eliminating exposure of sensitive devices to harsh reagents.
  • the analyte can exist in an impure form, i.e., mixed with one or more impurities.
  • the techniques of the described subject matter can be used to increase the PC of the analyte. Once the analyte is allowed to bind to the aptamers functionalized on the solid phases, the components are washed to remove any excess impurities. Analytes from another impure complex are allowed to bind to the aptamers, and another washing takes place. This procedure is repeated until the desired concentration is reached. The temperature is then set such that the concentrated analyte is released.
  • the described subject matter includes collection and detection components, for example, a surface tension-based microvalve for releasing the analyte onto a detection surface and a mass spectrometer used to measure the analyte.
  • detection techniques include detectors for measuring the amount of fluorescence given off by a sample of an analyte coupled with fluorescing materials, electrospray ionization mass spectroscopy, nuclear magnetic resonance, electrochemical techniques, impedance techniques, and the like.
  • the use of selective release of the analyte from the aptamers in a minimally invasive manner allows the aptamers to be reused. Minimal invasive release also causes less harm to the analyte.
  • the particular aptamer/analyte binding can cause an otherwise actively interactive analyte to be temporarily inactivated.
  • the analyte can be delivered to a target location where its interactivity is desired. Selective release can then release the analyte, which can regain the analyte' s original interactivity.
  • certain drugs are inactivated through aptamer binding and can be targeted to specific body locations for the drug to take effect.
  • Analytes include any appropriate biochemical component, biomolecule, pharmaceutical, protein, nucleotide sequence, cell, virus, compound, or the like.
  • analytes include toxic molecules, compounds, or bacteria, viruses, or the like, which can appear in pharmaceuticals, food, or the like.
  • aptamers can bind to peptides, proteins, small molecules, other inorganic and organic molecules, cells, viruses, micro organisms, and the like. It should be noted that analytes can be used beyond components merely for analytical purposes. Any suitable component which is selectively captured and released by an aptamer is encompassed within the described subject matter. For example, selective capture and targeted release of analytes can be used for drug delivery. Also, captured analytes can be permanently bound to an aptamer, such as in a technique for removing biochemical hazards from the environment.
  • analytes can be inactivated when attached to an aptamer, such as in techniques for reducing the effect of harmful chemicals. Still further, analytes can have their properties changed as a result of being bound to an aptamer, thereby producing a secondary effect of the analyte as desired.
  • the described subject matter includes a microfluidic device that accomplishes integrated, all-aqueous realization of specific extraction, concentration, and coupling to mass spectrometric detection of biomolecular analytes.
  • the device uses an aptamer (e.g., an oligonucleotide that binds specifically to an analyte via affinity interaction) functionalized on microbeads to achieve highly selective analyte capture and concentration.
  • an aptamer e.g., an oligonucleotide that binds specifically to an analyte via affinity interaction
  • the device makes novel use of thermally induced, reversible breakage of the analyte-aptamer complex at low temperature (38 0 C) to release the captured analyte and regenerate microbead surfaces.
  • a microfluidic device is used for PC and release of specific analytes.
  • the device surfaces are functionalized with an RNA aptamer that selectively binds a target analyte.
  • the device employs thermally induced denaturing of the aptamer for intelligent release. This occurs at 32.5 0 C, a safe temperature for thermally sensitive analytes and ligands functionalizing the device surface. Since denaturing the aptamer is reversible, this permits reuse.
  • the device includes a microchamber packed with aptamer-immobilized microbeads for analyte PC, a microheater and temperature sensor for thermally induced analyte release, and microchannels equipped with a passive valve using surface tension for spotting the released analyte onto a MALDI analysis plate (Fig. 14).
  • Analyte and wash solutions are introduced via a sample inlet 1400, while the matrix solution is introduced via a matrix inlet 1402.
  • the bead inlet facilitates packing a chamber 1404 with microbeads.
  • a resistive heater and sensor are placed below aptamer chamber 1404 to promote efficient heating and accurate sensing.
  • the valve and deposition well 1406 are placed near the aptamer chamber 1404 to reduce analyte dilution after release due to adsorption to the channel walls or diffusion to dead fluid volumes.
  • a waste valve 1408 is used to remove any excess fluids or impurities.
  • a heater 1410 is used to set the temperature of the chamber to an appropriate thermal release temperature.
  • the microfluidic chip structure is realized with three sandwiched polymer layers.
  • Layer 1412 incorporates the inlets, passive valve, and waste outlet.
  • layer 1414 provides an air vent connected to the spotting well. It also encapsulates the fluidic network present in layer 1412.
  • Layer 1416 defines the spotting well and houses an air vent channel. A vent 1418 is used to prevent dead air volume during spotting. The sample is deposited on to a MALDI plate 1420 for analysis.
  • PC is achieved on adenosine monophosphate (AMP) as a model analyte by use of an adenosine triphosphate aptamer (ATP-aptamer) on an integrated microfluidic device.
  • AMP adenosine monophosphate
  • ATP-aptamer adenosine triphosphate aptamer
  • MALDI-MS matrix assisted laser desorption/ionization mass spectrometry
  • AMP is introduced into the microchamber and extracted by the aptamer.
  • a rinse follows to flush out impurities through the waste outlet. For concentration, this procedure can be repeated to saturate AMP on the beads.
  • the microchamber is heated using the microheater to reverse the AMP/ATP-aptamer bond. This releases the analyte from the beads.
  • a valve based on surface tension is used. Passive microfluidic valves can employ surface tension for fluid regulation in low pressure devices.
  • a hydrophobic channel generates a pressure difference across the air-liquid interface occurring within it, governed by a modified Young-Laplace equation (which includes the pressure drop induced by channel geometry):
  • ⁇ , ⁇ c , w, and h are the surface energy, contact angle, width, and height of the channel, respectively, at the air- liquid interface.
  • This pressure drop allows the hydrophobic channel to act as a passive valve, and, in our device, is used to regulate flow between two zones. Since the packed chamber is the primary flow resister in zone 1 (gray channel network in Fig. 14), a modified Poiseuille equation is used to determine its pressure drop:
  • n, u, L, ⁇ , and d 0 represent the dynamic viscosity, average fluid velocity, channel length, void fraction, and bead diameter, respectively.
  • the pressure difference imparted by the passive valve separates flow within zones 1 and 2 (white channel network in Fig. 14).
  • the pressure drop in zone 1 is -220 Pa (Eq. 2).
  • the pressure drop in zone 1 must be greater than the valve's (1.7 kPA, Eq. 1). This is accomplished by plugging the waste outlet and maintaining a constant flow rate during deposition following thermally induced release of AMP from the aptamer. After sample spotting, the chip is removed from the MALDI plate for analysis.
  • Biotinylated ATP-aptamer is purified while AMP, cytidine, uridine, and guanosine triphosphate (C/U/G-TP) are synthesized.
  • the matrix solution is prepared from 2,4,6-trihydroxy-acetophenone (2,4,6-TH AP), 2,3,4- THAP, and diammonium citrate at 0.1, 0.05, and 0.075 M concentrations, respectively, in a 3:5 (v/v) mixture of acetonitrile/water.
  • Fig. 15 depicts an example fabrication process flow as seen from cross-section I-I in Fig. 14.
  • Fig. 15a depicts PR patterning for Cr/ Au deposition.
  • Fig. 15b depicts thermal evaporation of a Cr/ Au bi-layer.
  • Fig. 15c depicts lift-off patterning of Cr/ Au and PECVD deposition of SiO 2 .
  • Fig. 15d depicts a substrate drilled for fluidic ports and 3 through-hole polydimethylsiloxane (PDMS) layers aligned and permanently bonded.
  • Fig. 15e depicts a packaged chip with tubing.
  • PDMS polydimethylsiloxane
  • SU- 8 molds for each microfiuidic layer are first created, with which PDMS prepolymer is cast into an in-house built through-hole PDMS sandwiching jig and cured (60 °C for 8 hours). Meanwhile, Cr/ Au (5/100 nm) films are deposited, patterned, and passivated with SiO 2 on glass substrates, realizing the microheater and temperature sensor. Following plasma (O 2 ) treatment of each bonding interface, all three PDMS layers and the glass substrate are then aligned using optical microscopy and an x-y-z stage before permanently bonding them to each other consecutively. Finally, microbeads are packed into the aptamer chamber and the entire assembly is subsequently attached to a MALDI plate via spontaneous adhesion.
  • the device is first rinsed with water (10 ⁇ l/min, 10 min). All following washing and loading schemes are identical. ATP-aptamer is loaded (10 ⁇ M, 10 ⁇ l, 20 min) into the chamber to functionalize the bead bed. After a subsequent wash, a pure matrix mass spectrum (MS) is acquired for a negative control.
  • MS matrix mass spectrum
  • 0.1 and 1.0 ⁇ M AMP samples are loaded into the aptamer chamber separately. A rinse follows to eliminate non-specific compounds. AMP is then released from the aptamer by raising the chamber temperature to 38 °C while introducing a matrix sample plug. The sample/matrix plug is then transferred to the spotting well and deposited onto the MALDI plate to be subsequently analyzed. Similarly, for specific extraction of AMP, a solution of AMP, CTP, UTP, and GTP (1 ⁇ M) is loaded into the aptamer chamber. After an incubation (5 min) and wash procedure (to flush out non-target molecules), matrix is loaded into the chamber. The heater is activated to release the molecules currently on the aptamer and deposit them onto the MALDI plate for analysis.
  • a multiple injection scheme is used for preconcentration of AMP.
  • the aptamer chamber is consecutively loaded with 10 nM injections of AMP sample. Each injection is incubated (5 min) and followed by a rinse. Upon suspected saturation of the aptamer with AMP, the chamber is heated to release the analyte into a matrix plug. The analyte is then deposited for analysis.
  • a fluorescence solution is regulated between zones 1 and 2 (Fig. 16).
  • fluorescence solution bypasses the passive valve (Fig. 16a).
  • Fig. 16b Upon blocking the main outlet, a gradual increase in fluorescence through a valve 1602 to the spotting outlet (Fig. 16b) was observed.
  • Sample spot size can be a useful characteristic during MALDI analysis. Large volume spots can promote dissociation of matrix from sample upon spot crystallization, resulting in poor ionization.
  • Spot size produced by the device is measured as a function of driving flow rate (Fig. 17). For low flow rates (10-30 ⁇ l/min), spot sizes approximately equal to the well size are obtained ( ⁇ 500 ⁇ m). Higher flow rates (>40 ⁇ l/min) generate a larger spot diameter (-700-800 ⁇ m) since the seal between the PDMS and MALDI plate at the location of the spotting well tends to falter at the resulting higher pressures. Consequently, the sample spot broadens once the chip is removed from the plate to obtain a spot size. However, this is of no detriment to the overall performance of the device compared to conventional spotting (with syringe or pipette), where crystallized spots are larger (>1 mm).
  • AMP As described, to demonstrate AMP extraction by ATP-aptamer, two sample solutions of AMP (0.1 & 1.0 ⁇ M) are first injected into the chamber. AMP is released and deposited onto a MALDI-MS plate and analyzed (Fig. 18). The MS of a spot obtained from a 0.1 ⁇ M AMP solution (Fig. 18a) shows a distinctive mass peak of 348 Da, which corresponds to AMP (established value: 347.22 Da). Since AMP concentration is relatively low, the magnitude of this peak is comparable to several peaks from the MALDI matrix (338, 392, 468 & 502 Da). A mass spectrum obtained from a 1.0 ⁇ M AMP solution (Fig. 18b) improves the analyte-to-reference peak contrast. In this case, the AMP peak dominates reference peak amplitudes, suggesting that concentrating dilute samples can improve analyte recognition.
  • purification of analytes can be a valuable tool for selectively controlling analytes in biochemical applications.
  • AMP is selectively extracted from a homogeneous solution of AMP, CTP, UTP, and GTP (1.0 ⁇ M each) by loading the sample into the aptamer chamber and subsequently washing the chamber to isolate AMP.
  • a deposited sample spot is obtained similarly to previous protocol.
  • Fig. 19 represents the MS of an analyte sample originating from the homogeneous solution. The ratio of AMP to noise is comparable to that seen in Fig. 18b, where only AMP is present in the solution. Additional non-target peaks are observed (483, 484, & 523 Da). However, their intensities are significantly lower than the AMP peak, suggesting that the amount of non-specific binding is negligible. This confirms the ability of the described subject matter to selectively extract and concentrate biomolecules for analytical applications.
  • PC can be useful for sample conditioning and analyte signal improvement.
  • PC performance of the device is demonstrated by loading a dilute AMP sample into the aptamer chamber multiple times to saturate the analyte on the aptamer bed before release for MS analysis.
  • Dilute sample concentration is chosen to be lower (-0.01 ⁇ M) in order to highlight the detection enhancement due to PC.
  • An MS is obtained from the resulting sample spot (Fig. 20a).
  • An AMP peak to noise ratio slightly higher than that seen in Fig. 18a is observed, demonstrating the successful concentration of AMP by ⁇ 1 Ox.
  • EXAMPLE 2 Another embodiment illustrates the principles of the described subject matter.
  • Biotinylated adenosine triphosphate aptamer (bio-ATP-40-1, or ATP- aptamer) is HPLC purified by Integrated DNA Tech. AMP is synthesized and fluorescently labeled with thiazole orange (TO).
  • Buffer solution (pH 7.4) is prepared from Tris-HCl (20 mM), NaCl (140 mM), KCl (5 niM), and MgCl 2 (5 niM) in water.
  • Streptavidin coated polystyrene beads (50-80 ⁇ m, OD) are acquired from Pierce.jjack: pierce?]
  • a Nikon Eclipse TE300 microscope and CCD is employed for fluorescence detection. Temperature control is accomplished with a thermoelectric device and type-K thermocouple.
  • a New Era NE- 1000 syringe pump enables flow in the device.
  • FIG. 1 A device schematic is shown in Fig. 1.
  • Channels 100 and 102 A device schematic is shown in Fig. 1.
  • Microbead 114 packing into the chamber is accomplished through 104.
  • Ports 106, 108, and 110 are 1 mm in radius and 140 ⁇ m thick.
  • Chamber and microfluidic network volumes, respectively, are 3.09 ⁇ l and 3.60 ⁇ l.
  • Channels are fabricated using PDMS micro-molding by soft lithography (Fig. 2).
  • a mold is created on a 4-in silicon wafer by patterning SU-8.
  • PDMS pre-polymer solution is mixed (10:1; w.w), degassed, and semi-cured (70 0 C, 50 min) over the mold (Fig. 2b).
  • glass substrates are cleaved (25 mm x 30 mm) and drilled to create ports 106-110 (Fig. 2c).
  • the semi-cured PDMS sheet is removed from the mold, aligned, and bonded to the glass following O 2 plasma treatment of the bonding interface. Permanent bonding is realized with a final bake (25 min at 85 0 C).
  • Packaging of the device is accomplished by inserting silica capillary and Tygon tubing (Fig. 2d), (0.6 mm ID, 0.7 mm OD) and (0.6 mm ID, 3.18 OD), respectively into ports 106-110. The interfaces are then sealed with epoxy.
  • the device is mounted on the microscope stage using clips or double- sided tape.
  • a blue excitation filter combined with a green-pass dichroic mirror is used.
  • a 10x objective is kept focused on a single area of the chamber.
  • the chamber is initially rinsed with buffer (50 ⁇ l/min, 10 min). The following rinses are identical. Streptavidin coated beads are introduced via c3 by manual pressure. The chamber and channels are rinsed and bio-ATP-aptamer is injected (20 ⁇ M, 20 ⁇ l, 10 ⁇ l/min) and incubated (20 min) in the chamber. After a final rinse, a fluorescence control is established. Extracting distinct concentrations of AMP (24.5 0 C, 10 ⁇ l, 10 ⁇ l/min) establishes a fluorescence intensity curve. The procedures use the above injection parameters. Solution concentrations range from 0.1-10 ⁇ M and fluorescence is detected after rinsing between separate extractions. For PC of AMP, multiple solution injections are used. Two devices
  • AMP solution is extracted at increasing concentrations onto multiple devices. A roughly linear increase in signal intensity is observed as AMP concentration in solution increased (Fig. 3).
  • Adenosine triphosphate is purchased from Sigma-Aldrich Co. (Milwaukee, WI). Diethyl pyrocarbonate treated sterile water (SW), from Fisher (Pittsburgh, PA), is used. Buffer solution (pH 7.4) is prepared by mixing Tris-HCl (20 mM), NaCl (140 mM), KCl (5 mM), and MgCl 2 (5 mM) in sterile water. Chemicals for the buffer solution are purchased through Fisher Scientific.
  • ATP aptamer, TO-AMP, and ATP working solutions are all prepared using Tris-HCl buffer.
  • UltraLink immobilized streptavidin polystyrene beads (50-80 ⁇ m in diameter) are acquired from Pierce (Rockford, IL). All solvents, isopropyl alcohol (IPA), methyl alcohol, and acetone are of purified grade (Mallinekrodt Baker, Phillipsburg, NJ).
  • SU-8 2025 and 2100 is purchased from MicroChem (Newton, MA).
  • Poly-dimethylsiloxane (PDMS) is acquired from Robert McKeown Company (Somerville, NJ). Torr Seal epoxy and silicone glue is obtained from Varian (Palo Alto, CA) and Action Electronics (Santa Ana, CA), respectively.
  • Glass slides 25 mm x 75 mm are purchased from Fisher. [JACK: Fisher?] Silica capillary tubing and Tygon poly-vinyl chloride (PVC) tubing are purchased from Polymicro Technologies (Phoenix, AZ) and McMaster Carr (Dayton, NJ), respectively.
  • Arctic Silver 5 is obtained from Arctic Silver Inc. (e.g., used for IC component bonding) and Kapton Tape is purchased from Techni- Tool (Worcester, PA).
  • Microfluidic flow is provided from a New Era model NE- 1000 syringe pump (Farmingdale, NY), 5 cm 3 syringes, and 21 gauge (38.1 mm long) needles (Becton Dickinson, Franklin Lakes, NJ). Diamond- tipped drill bits (0.7 mm diameter) and a Model 7000 standard drill press are obtained from Servo Products (Eastlake, OH).
  • a device is shown in Fig. 8.
  • the channels are numbered for reference.
  • Channels 800 and 802 (5.1 mm x 400 ⁇ m x 40 ⁇ m) are used to deliver sample and buffer solution to the chamber (8.7 mm x 3 mm x 140 ⁇ m).
  • Channel 804 is used to pack the beads 814 (e.g., polystyrene beads).
  • the ports have radii of 1 mm each and are 140 ⁇ m thick.
  • chamber 812 has an effective volume of 3.09 micro-liters with the tapers taken into consideration, whereas the microfluidic device volume (on- chip) is 3.60 micro-liters.
  • Poiseuille-flow the maximum pressure drop across this device (port to port), excluding beads, can be calculated from:
  • Q is the flow rate
  • Ap is the pressure drop
  • ⁇ is the dynamic viscosity of the fluid
  • / is the channel length
  • D ⁇ is the hydraulic diameter given by the expression
  • A is the cross-sectional area of the channel and P is the wetted perimeter.
  • Q 50 ⁇ l/min used in the demonstrations is 6.83 kilo-Pascal.
  • the pressure increase is estimated to be 10-20 times greater.
  • SPE microchip solid-phase extraction
  • Fabrication begins with deposition and patterning of 15 nm Cr alignment marks via thermal evaporation on an Edwards/BOC Auto306 thermal evaporator (Wilmington, MA), followed by lift-off in acetone overnight. Secondly, patterning of SU-8 2025 realizes channels 800 and 802 (40 ⁇ m thick) (Fig. 9b), whereas SU-8 2100 resist completes the mold, producing the reaction chamber and channel 804 (140 ⁇ m).
  • PDMS pre-polymer solution is mixed with a mass ratio of 10:1 and distributed on the mold (Fig. 9c).
  • the pre-polymer is degassed by vacuum (30 min) and followed by semi-curing (70 °C, 50 min).
  • glass substrates are diced (25 mm x 30 mm) and drilled to create the access ports (806-810) (Fig. 9d).
  • the glass substrates are then cleaned using a solution of H 2 SO 4 and H 2 O 2 (7:4 vol/vol at 130 0 C).
  • ports can be fabricated in the PDMS blank layer.
  • the semi-cured PDMS sheet is removed from the SU-8 mold, aligned and bonded to the glass slides following O 2 plasma treatment of the bonding interface in a Technics Series 800 Micro RlE device (100 mtorr and 85 W) for 15 seconds. Permanent bonding and curing of PDMS to the substrate is performed by heating the chip (25 min at 85 0 C).
  • Packaging is accomplished by inserting a combination of silica capillary tubing (0.6 mm ID, 0.7 mm OD) segments along with Tygon PVC tubing (0.6 mm ID, 3.18 OD) through the drilled access ports (Fig. 9e).
  • the connection interfaces are sealed using silicone glue and Torr seal epoxy.
  • a thermocouple is subsequently sandwiched between a peltier device and the bottom of the microfluidic chip (Fig. 9f). The components are held together by thermal interfacing paste or Kapton Tape.
  • Fluorescence detection is done using a Nikon TE300 and a Q-Imaging Retiga 2000R device. During analyte binding, TO emission occurs at 530 nm when excited at 480 nm, so a blue light filter and green-pass dichroic mirror are used accordingly. The device is mounted in the same position using double-sided scotch tape marks on the microscope stage. For each image, a 10 ⁇ objective is used to collect emitted fluorophores from the same area of the chamber (Fig. 10). These operating conditions are identical for all images taken for fluorescence detection of the analyte.
  • the entire microfiuidic device is flushed (50 ⁇ l/min) with the buffer solution for 30 minutes by using any port as an inlet and collecting waste from both remaining ports.
  • Streptavidin coated beads are suspended in buffer (4 ml) and loaded into a 5 ml syringe. Manual pressure is used to pack the beads from channel 804 via port 810 into the chamber. Subsequently, channel 804 is sealed permanently near the port interface using silicone glue.
  • the chamber and channels are washed (50 ⁇ l/min) with buffer (30 min) through 800.
  • ATP-aptamer solution (20 ⁇ l, 20 ⁇ M) is injected (10 ⁇ l/min) and allowed to incubate (20 min) in the chamber. The channel is washed again (50 ⁇ l/min for 20 min) and a baseline fluorescence signal is taken.
  • TO-AMP at different concentrations 400 nM, 500 nM and 10 ⁇ M
  • the solution is kept stagnant in the chamber for 10-15 minutes to allow complete interaction between the analyte and aptamer surface of the beads.
  • the chamber is washed (50 ⁇ l/min for 15 min) with buffer to eliminate all non-specific compounds, un-reacted molecules, and impurities. A subsequent fluorescence image is taken.
  • TO-AMP is released and collected in two ways: the first technique uses competitive displacement of TO-AMP by incubating different concentrations of ATP (800 ⁇ M and 3200 ⁇ M); the second technique uses elevated chip temperature (80 0 C) while buffer is flowed (10 ⁇ l at 5 ⁇ l/min) through to collect analyte. During purification, time resolved analyte adsorption demonstrations are conducted. For a 400 nano-molar concentration of TO-AMP solution, fluorescence micrographs are recorded at time intervals of 1 minute. Images ceased to be taken after the observed fluorescence level shows no appreciable change.
  • An integrated SPE bed is prepared using a double weir design forming a cavity.
  • PDMS for the channel material can present challenges. Since PDMS is pliable, beads can be pushed under the weir structures under positive pressure resulting in backpressure. During bead introduction, port 810 is prone to clogging. Designs using drilled access ports in the PDMS blank layer can be a source of this problem.
  • the holes in PDMS are plagued with burrs containing loose PDMS particles not cleared while drilling, which became obstacles and instigated clogging. This is mitigated by generating reversed flow and allowing beads to dislodge and flow back toward the source. On occasion, several forward/reverse pumping cycles are used to fully clear obstructions and continue filling the chamber. Drilling holes in the glass slides provides smoother, burr-free edges. Using this technique, fully packed chambers are realized in over 90 percent of devices.
  • the device is capable of capturing and releasing TO-AMP using two release techniques.
  • the first is competitive displacement using a concentration gradient of ATP analyte (Fig. 13a).
  • the second is thermal energy (Fig. 13b).
  • Fig. 13b After each competitive ATP solution (800 ⁇ M and 3.2 raM) injection (10 ⁇ l), fluorescence intensity is measured. Five extraction and release cycles are performed (2 shown here) using thermal energy. Solution conditions and sampling using TO-AMP (400 nM) closely tracks those employed in SPE demonstrations. Fluorescence measurements are taken after each capture and release wash procedure.
  • the temperature control can be used to either raise or lower the temperature.
  • a resistor in contact with the micro chamber where the analyte/aptamer complex is located can be used to raise the temperature.
  • a cooling mechanism such as an air cooler, refrigerant mechanism, or the like can be used to lower the temperature of the analyte/aptamer complex.
  • the specific set point at which the aptamer/analyte bond is released can either be above the temperature of the device (e.g., the temperature raised using a heater) or can be lower than the temperature of the device (e.g., the temperature lowered using a cooling mechanism).
  • the specific parameters and needs of the application can dictate the specific temperature shift needed for thermal release of the analyte.

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des techniques et des composants pour la capture et la libération sélectives d'invasion minimale d'analytes. Un aptamère est sélectionné pour son affinité de liaison avec un ou des analytes particuliers. L'aptamère est fonctionnalisé sur une phase solide, par exemple des microbilles, un monolithe de polymère, une phase solide microfabriquée, etc. [INVENTEURS : MERCI DE SPÉCIFIER SI D'AUTRES PHASES SOLIDES SONT POSSIBLES]. L'analyte est autorisé à se lier à l'aptamère, par exemple dans une microchambre. Une fois que l'analyte a été lié, une commande de température fixe la température à une température appropriée à laquelle l'analyte capturé est libéré.
PCT/US2008/058433 2007-03-27 2008-03-27 Capture et libération sélectives d'analytes Ceased WO2008119018A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US12/568,651 US20100151465A1 (en) 2008-03-27 2009-09-28 Selective Capture and Release of Analytes
US13/652,214 US9250169B2 (en) 2007-03-27 2012-10-15 Selective capture and release of analytes
US14/978,716 US20160169780A1 (en) 2007-03-27 2015-12-22 Selective capture and release of analytes

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US90830407P 2007-03-27 2007-03-27
US90829807P 2007-03-27 2007-03-27
US60/908,298 2007-03-27
US60/908,304 2007-03-27
US96880307P 2007-08-29 2007-08-29
US60/968,803 2007-08-29
US97206107P 2007-09-13 2007-09-13
US60/972,061 2007-09-13
US98747407P 2007-11-13 2007-11-13
US60/987,474 2007-11-13
US98918207P 2007-11-20 2007-11-20
US60/989,182 2007-11-20

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060172429A1 (en) * 2005-01-31 2006-08-03 Nilsson Erik J Methods of identification of biomarkers with mass spectrometry techniques
US20060205061A1 (en) * 2004-11-24 2006-09-14 California Institute Of Technology Biosensors based upon actuated desorption

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060205061A1 (en) * 2004-11-24 2006-09-14 California Institute Of Technology Biosensors based upon actuated desorption
US20060172429A1 (en) * 2005-01-31 2006-08-03 Nilsson Erik J Methods of identification of biomarkers with mass spectrometry techniques

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