Patent Application
20170292950
Embodiments of the present disclosure relate generally to devices and methods of detecting bacteria using polymer materials.
Molecularly imprinted polymers (MIPs) can be used for detecting chemical substances in complex mixtures. In modern research, these polymers are of increasing interest for bioanalytical applications. Advantages of using these MIPs include easy and cheap production; mechanical, chemical, and thermal stability; reusability; and long shelf life. In recent years, the concept of molecular imprinting has been extended to surface imprinting of thin polymer films with micrometer-sized cells to create so-called “surface imprinted polymers” (SIPs) for the detection of proteins, glycoproteins, plant viruses, human viruses, bacteria, pollen, yeast cells, and even mammalian red blood cells. SIPs are polymeric materials with indentations at the surface, with a form and function matching part of a desired target. SIPs are suitable for bonding with larger objects (e.g., cells, bacteria, etc.), which do not diffuse quickly through pores of an MIP. Imprinting may occur after polymerization by softening the polymer. The detection of cells using biosensors described in literature is conventionally done by gravimetric detection, electronic read-out platforms or micro-fluidic techniques. However, these techniques are often time-consuming, provide difficulties for analysis, or require expensive equipment.
For example, temperature resistance of substrates having MIPs attached thereto based on the concentration of analytes is described in U.S. Patent Application Publication 2014/0011198 A1, “Heat-Transfer Resistance Based Analysis Bioparticles,” published Jan. 9, 2014, the entire disclosure of which is hereby incorporated herein by reference.
A low-cost sensor platform providing the capability to differentiate between cells with slight differences in shape, size, and functionalities in functional groups on their surface would be a valuable tool for modern research and industry.
In some embodiments, a method for characterizing bacteria includes passing a liquid containing an analyte comprising a first bacteria and a second bacteria over and in contact with a polymer material on a substrate. The polymer material is formulated to bind to the first bacteria, and the first bacteria binds to the polymer material with a higher affinity than the second bacteria. A heat transfer property of the polymer material varies based on an amount of the analyte bound thereto. The method further includes binding a portion of the first bacteria and the second bacteria of the analyte to the polymer material, removing at least a portion of the second bacteria from the polymer material, detecting a temperature of the substrate, and calculating a concentration of the first bacteria in the liquid based at least in part on the temperature of the substrate.
In other embodiments, a method for characterizing a liquid comprising bacteria includes passing a liquid containing a first strain of bacteria and at least a second strain of bacteria over and in contact with a polymer material on a substrate. The polymer material is formulated to bind to the first strain of bacteria, and the first bacteria binds to the polymer material with a higher affinity than the at least a second bacteria. A heat transfer property of the polymer material varies based on an amount of material bound thereto. The method further includes binding a portion of the first bacteria and a portion of the at least a second bacteria to the polymer material, washing the polymer material to remove the at least a second bacteria therefrom, passing the liquid over the polymer material after washing the polymer material, washing the polymer material at least a second time to remove the at least a second bacteria therefrom, detecting a temperature of the substrate, and calculating a concentration of the first bacteria in the liquid based at least in part on the temperature of the polymer material.
The illustrations presented herein are not actual views of any particular device or method, but are merely idealized representations employed to describe example embodiments of the present disclosure. Elements common between figures may retain the same numerical designation.
As used herein, the terms “template molecule” and “template bacteria” respectively refer to molecules or bacteria used to form a molecularly imprinted polymer (MIP) or surface imprinted polymer (SIP). Such MIPs or SIPs can then detect “target molecules” or “binding partners,” which have functionality corresponding to the template molecules used to form the MIP or SIP.
As used herein, the term “may” encompasses the word “can,” and the term “may be” encompasses the words “is” or “are,” depending on context. Furthermore, presence of the word “may” is intended to indicate options for practicing or implementing embodiments of the disclosure, without limitation.
The device 100 may include a substrate 110 having a polymer material 112 located over a surface thereof. For example, the polymer material 112 may be formed or disposed over a generally planar surface of the substrate 110, and another, opposite generally planar surface of the substrate 110 may be free of the polymer material 112. In some embodiments, the substrate 110 may include a metal (e.g., aluminum), an alloy, a semiconductor (e.g., silicon, doped diamond, etc.), an electrically insulating material (e.g., undoped diamond). The polymer material 112 may include any material for which a heat transfer property varies based on an amount of the analyte bound thereto. For example, the thermal conductivity, thermal diffusivity, heat capacity, or another property of the polymer material 112 may vary with concentration of the analyte on the surface thereof.
In some embodiments, the polymer material 112 may include an imprinted polymer, such as a molecularly imprinted polymer (MIP) or a surface imprinted polymer (SIP). MIPs and SIPs may also be referred to in the art as “plastic” antibodies. MIPs typically possess a high affinity for a specific binding partner, so that when such binding partners are contacted with the MIP, the molecules bind with the MIP. MIPs are synthetic receptors that contain nanocavities with high affinity for their respective target molecules. Imprinting (i.e., formation of the nanocavities) is often part of the polymerization process. MIPs are able to specifically bind targets, including bacteria, varying from small ions to large cells in complex matrices. Binding of molecules to the MIP may alter some properties of the MIP, such as thermal properties, mechanical properties, electrical properties, etc. The altered property of an MIP may, therefore, be used to detect a presence of such molecules at relatively low concentrations. MIPs are described in, for example, U.S. Patent Application Publication 2009/0281272 A1, “Monodisperse Molecularly Imprinted Polymer Beads,” published Nov. 12, 2009, the entire disclosure of which is hereby incorporated herein by reference.
Similarly, SIPs typically possess a high affinity for a specific binding partner, but may typically bind to relatively larger objects (e.g., cells, bacteria, etc.) that do not diffuse quickly through pores of an MIP. SIPs may be polymer materials formed over a surface, then imprinted after polymerization by softening the polymer.
In certain embodiments, the polymer material 112 may include DNA, RNA, proteins, or portions or analogs thereof. For example, the device 100 may include a substrate 110 (e.g., a diamond surface) functionalized with a polymer material 112 such as DNA, RNA, a protein, a polypeptide, a nucleic acid polymer, a probe, or a portion or analog thereof (e.g., complementary DNA, antibodies, etc.). The polymer material 112 may be formulated to possess a high affinity for a specific binding partner, so that when such binding partners are contacted with the surface of the substrate 110, the molecules bind with the polymer material 112. The polymer material 112 may also bind to analogues of the binding partner (e.g., a material having similar functionality as the binding partner), though not necessarily with the same affinity as binding with the binding partner itself. In some embodiments, the polymer material 112 may include at least about seven (7) repeating units, such as ten (10) repeating units or more.
In some embodiments, the polymer material 112 may include a material screen-printed onto the substrate 110. Screen-printed materials may be manufactured efficiently and in mass quantities, with relatively high uniformity in comparison with other materials.
The device 100 may further include a heat sink 114 thermally coupled to a surface of the substrate 110, such as a surface opposite the polymer material 112. Though referred to as a heat “sink” for the sake of simplicity, the heat sink 114 may be configured to provide heat to or remove heat from the substrate 110 and, so, may also be characterized as a heat transfer element 114. The heat sink or heat transfer element 114 may be a material having a high thermal conductivity, such as a transition metal (e.g., copper, silver, etc.) or an alloy or mixture thereof. In some embodiments, the polymer material 112 may be applied to the heat sink 114 itself. The heat sink 114 may be thermally coupled to a temperature sensor 116 (e.g., a thermocouple or another device) configured to detect a temperature of the heat sink 114, and to a temperature modification device 118 configured to maintain the temperature of the heat sink 114. The temperature modification device 118 may include, for example, a thermoelectric device, a heat exchanger, a fan, a resistance heater, etc. The temperature sensor 116 may be a resistor having a resistance that varies with temperature. If the properties of the heat sink 114 are known (e.g., if a relationship between a control signal to the modification device 118 and the temperature of the heat sink 114 is well characterized), the temperature sensor 116 may be omitted. In some embodiments, the temperature sensor 116 may be integral to the temperature modification device 118. For example, the internal resistance of the temperature modification device 118 itself may be measured to determine its temperature.
The temperature sensor 116 and the temperature modification device 118 may be connected to a controller 121 configured (i.e., programmed) to control the temperature modification device 118 to cause the heat sink 114 to produce a thermal wave emanating from the heat sink 114 and through the substrate 110 (including the polymer material 112 thereon). For example, the controller 121 and a processor 123 may be incorporated into a computer 120 (e.g., the controller 121 may be an input-output card configured to receive and provide electrical signals, and may be configured to receive signals from the processor 123). In some embodiments, the controller 121 may be a proportional-integral-derivative (PID) controller capable of changing the temperature of the heat sink 114 by a small amount on a relatively short time scale. For example, the controller 121 may change the temperature of the heat sink 114 by about 0.5° C. or less, about 0.2° C. or less, or even about 0.05° C. or less. Thus, the thermal wave may have an amplitude of about 1.0° C. or less, about 0.4° C. or less, or even about 0.10° C. or less. The controller 121 may be capable of changing the temperature of the heat sink 114 via the temperature modification device 118 from one set point to another and back to form a thermal wave having a frequency from about 0.001 to about 0.5 Hz, such as from about 0.005 to about 0.1 Hz, or from about 0.01 to about 0.05 Hz. In some embodiments, the controller 121, the temperature modification device 118, and the heat sink 114 may together produce a thermal wave having a variable frequency. Based on a measurement from the temperature sensor 116 (if present), a known input to the temperature modification device 118, or other means, properties of the thermal wave may be known (e.g., a phase, amplitude, frequency at a specific time, rate of frequency change, etc.).
In other embodiments, the controller 121 may be configured to maintain the heat sink 114 at a constant temperature. Detection of analytes using a heat sink at constant temperature is described in U.S. Patent Application Publication 2015/0219584 A1, “Biosensor Using Impedimentric Real-Time Monitoring,” published Aug. 6, 2015, the entire disclosure of which is hereby incorporated herein by reference.
The device 100 may further include a flow cell 122 configured to pass a liquid 124 over the polymer material 112 of the substrate 110. The flow cell 122 may define a void 126 adjacent the polymer material 112 of the substrate 110, as well as an inlet 128 and an outlet 130 through which the liquid 124 may flow. An O-ring 131 or another appropriate sealing mechanism may retain the liquid 124 within the flow cell 122 adjacent the polymer material 112 over the substrate 110.
The liquid 124 may include an analyte 132, such as one or more strains of bacteria. The analyte 132 (which may include multiple analytes 132a and 132b) may specifically bind to the polymer material 112 and changes thermal properties thereof, as described above. If multiple analytes 132a and 132b are present in the liquid 124, the analytes 132a, 132b may have similar functionalities, such that each of the analytes 132a, 132b bind to the polymer material 112. The analytes 132a, 132b may bind to the polymer material 112 with different affinities. In some embodiments, the first analyte 132a may include living bacteria, and the second analyte 132b may include dead bacteria of the same species. In other embodiments, the first analyte 132a may include bacteria, and the second analyte 132b may include an analogue bacteria.
A temperature sensor 134 (e.g., a thermocouple or another device) may be configured to detect a temperature of the liquid 124 in (e.g., flowing through) the flow cell 122. The computer 120 may record the temperature of the liquid 124 by, for example, measuring a resistance of the temperature sensor 134 via the controller 121 and/or the processor 123, and correlating that resistance to a temperature. The temperature of the liquid 124 may be different from the temperature of the heat sink 114, and may vary based at least in part on the presence or absence of the analyte 132 and its concentration in the liquid 124. For example, temperature resistance of substrates based on the concentration of analytes is described in U.S. Patent Application Publication 2014/0011198 A1, “Heat-Transfer Resistance Based Analysis Bioparticles,” published Jan. 9, 2014, the entire disclosure of which is hereby incorporated herein by reference.
In some embodiments, the processor 123 may be configured to calculate a concentration of the analyte 132 in the liquid 124 based at least in part on a phase shift between the thermal wave produced by the heat sink 114 and an attenuated thermal wave in the liquid 124 after the thermal wave passes through the substrate 110 and the polymer material 112.
In other embodiments, the processor 123 may be configured to calculate a concentration of the analyte 132 based on a steady-state temperature difference between the heat sink 114 and the liquid 124.
In certain embodiments, the analyte 132 may bind to a non-planar surface. For example,
The base material 212 may be a polymer material such as polylactic-(L)-acid, which may be referred to in the art as PLLA. PLLA is transparent, inexpensive to produce from environmentally renewable sources (e.g., starch or sugar-containing agricultural products), biodegradable, and biocompatible. Furthermore, PLLA can be solubilized in chloroform to enable application to the thermocouple 210. Another material, rather than PLLA, may be selected to be the base material 212, based on desired properties. In some embodiments, the base material 212 may include polyurethane, polylactic acid, polycaprolactone, poly(lactic-co-glycolic acid), poly(D,L-lactide-co-glycolide), or another selected polymer. The base material 212 may be in the form of a thin, smooth, and homogeneous coating over the exterior of the thermocouple 210. Uniformity of the coating by base material 212 may enable to the device 200 to yield reproducible results. The thickness of the base material 212 may be selected in view of the thermal resistance of the base material 212 to affect the rate at which heat may flow toward or away from the thermocouple 210. Thus, a thinner base material 212 may be beneficial for applications in which a fast response is desired or temperature differentials are small.
The base material 212 may be selected to exhibit at least some elasticity, such that the device 200 may be flexible to allow bending of the thermocouple 210 without breaking the base material 212. This may enable the device 200 to be used for applications requiring tight clearance or bends (e.g., in vivo use in catheters).
An assay polymer 214 may be on a surface of the base material 212. In some embodiments, the assay polymer 214 may be directly bonded to the surface of the thermocouple 210, and the base material 212 may be omitted. The assay polymer 214 may include a material for which a heat transfer property varies responsive to an amount of the analyte bound thereto. For example, the thermal conductivity, thermal diffusivity, heat capacity, or another property of the assay polymer 214 may vary with concentration of the analyte on the surface thereof.
In some embodiments, the assay polymer 214 may include an imprinted polymer (an MIP or SIP), DNA, RNA, proteins, or portions or analogs thereof (e.g., antibodies). The assay polymer 214 may be configured to possess a high affinity for a specific binding partner, so that when such binding partners are contacted with the surface of the thermocouple 210, the molecules bind with the assay polymer 214. In some embodiments, the assay polymer 214 may include at least about seven (7) repeating units, such as ten (10) repeating units or more.
In some embodiments, the device 200 may include a processor 223 programmed to calculate an amount of the analyte bound to the assay polymer 214. The processor 223 may calculate a concentration of the analyte in a liquid in contact with the device 200 based at least in part on the amount of the analyte bound to the assay polymer 214. For example, the processor 223 may calculate the amount of the analyte by a method as disclosed in U.S. Patent Application Publication 2014/0011198 A1, “Heat-Transfer Resistance Based Analysis Bioparticles,” published Jan. 9, 2014; or U.S. Patent Application Publication 2014/0242605 A1, “Heat-Transfer Resistance Based Analysis of Bioparticles,” published Aug. 28, 2014, the entire disclosures of each of which are hereby incorporated herein by reference. In certain embodiments, the processor 223 may be used to detect a phase shift between a thermal wave at or emanating from a heat sink and an attenuated thermal wave at the thermocouple 210. The processor 223 may then calculate the concentration of the analyte in the liquid based at least in part on a difference in amplitude between the thermal wave at the heat sink and the attenuated thermal wave at the thermocouple 210.
Returning again to
The heat sink 114 may be thermally coupled to the substrate 110 at a surface opposite the polymer material 112. For example, the heat sink 114 may be placed in direct physical contact with the substrate 110 such that heat can flow from the heat sink 114 to the substrate 110 by conduction. In some embodiments, a thermally conductive material (e.g., a polymerizable liquid matrix having a thermally conductive filler) may be placed in physical contact with the heat sink 114 and the substrate 110 to eliminate air gaps between the heat sink 114 and the substrate 110. Similarly, the temperature modification device 118 may be thermally coupled to the heat sink 114 by direct physical contact, through a thermally conductive material, or by other appropriate means.
The flow cell 122 may be secured adjacent the substrate 110 such that the liquid 124 enters the flow cell 122 through the inlet 128, contacts the polymer material 112, and then leaves the flow cell 122 through the outlet 130. In some embodiments, the flow cell 122 may be connected to the heat sink 114 by one or more fasteners 138 (e.g., screws). In other embodiments, the flow cell 122 may be connected to the heat sink 114 by integral threads or by a slip-fit joint. The O-ring 131 or other seal may be configured to keep the liquid 124 from contacting the heat sink 114, the temperature modification device 118, or the back side of the substrate 110 directly.
The temperature sensor 134 may be disposed within the void 126 of the flow cell 122 to measure the temperature of the liquid 124 flowing through the flow cell 122. The temperature sensor 134 may be secured to the flow cell 122 by an adhesive or other appropriate means. The temperature sensor 134 may be electrically connected to the processor 123, which may include an ohmmeter. The processor 123 may be configured to continuously detect the temperature at the temperature sensor 134, and to calculate the concentration of the analyte 132 in the liquid 124.
The device 100 shown in
To detect the analyte 132, the liquid 124 containing the analyte 132 may be passed through the flow cell 122, adjacent and in contact with the polymer material 112 over the substrate 110. The analyte 132 (e.g., bacteria) binds to the polymer material 112, changing one or more thermal properties of the polymer material 112. The liquid 124 may flow continuously through the flow cell 122 during detection, or the flow may terminate before detection begins. In some embodiments, the flow cell 122 and the liquid 124 therein may be brought to a test temperature before detection of the analyte 132. As discussed above, the polymer material 112 may be a molecularly imprinted polymer formulated to bind a particular analyte 132 of interest.
In some embodiments, the first analyte 132a may be distinguished from the second analyte 132b by removing the second analyte 132b from the polymer material 112. For example, if the first analyte 132a is living bacteria, and the second analyte 132b is dead bacteria, the dead bacteria may be washed or rinsed from polymer material 112 (e.g., with a buffer), leaving the living bacteria behind. Differences in affinity between the first analyte 132a and the second analyte 132b may facilitate such discrimination. In some embodiments, the first analyte 132a may be the template molecule used to form the polymer material 112, and the second analyte 132b may be a molecule or bacteria having some similar functionality. Therefore the second analyte 132b may bind, at least weakly, to the polymer material 112.
Characterized strains of Escherichia coli (ATCC® 8739™) and Staphylococcus aureus (ATCC® 6538™) were obtained from Leibniz Institute DSMZ, of Braunschweig, Germany. 20 ml of nutrient broth (item number x929.1, from Carl Roth GmbH+Co KG, of Karlsruhe, Germany) was inoculated with a single colony of E. coli. 20 ml of Caso broth (item number x938.1, from Carl Roth) was inoculated with a single colony of S. aureus. Both colonies were allowed to grow overnight at 37° C. while subject to agitation.
1 ml of each overnight culture was diluted in 20 ml of the respective broth, and allowed to grow at 37° C. for 3 hours or until OD600 (i.e., optical density measured at a wavelength of 600 nm, a measurement correlated to concentration of the bacteria) of 1 was obtained. Afterwards, the cells were harvested by centrifuging to form pellets, which were washed one time with phosphate buffered saline (PBS), and then resuspended in PBS to achieve desired concentrations.
A spin-coating solution was prepared by dissolving 122 mg of 4,4′-diisocyanatodiphenylmethane, 222 mg of bisphenol A, and 25 mg of phloroglucinol in 500 μL of anhydrous tetrahydrofuran (THF). All reagents had a purity of at least 99.9% and were used as received from Sigma-Aldrich N.V., of Diegem, Belgium. The solution was polymerized up to its gel point at 65° C. for 200 minutes while gently stirring. The solution was diluted in anhydrous THF in a 1:5 ratio. Polyurethane layers with an average thickness of 1.2±0.1 μm, as measured with a profilometer (Dektak 3ST, Sloan Instruments Corporation, Santa Barbara, Calif., USA) were formed by spin-coating the solution for 60 s at 2000 rpm onto aluminum substrates each having a surface area of 1 cm2.
Polydimethylsiloxane (PDMS) stamps were made using a Dow Corning SYLGARD® 184 silicone elastomer kit purchased from Malvom N.V., of Schelle, Belgium. Bacteria-covered PDMS stamps were formed by applying 400 μL of a bacteria suspension in PBS to each stamp. The bacteria were allowed to settle to the surface of the stamp for 60 s. The excess fluid was removed by spin-coating the stamps at 3000 rpm for 60 s to create a dense monolayer of bacteria on the stamp surface.
The bacteria-covered stamps were each pressed into the polyurethane layer on one of the aluminum substrates at a pressure of 70 Pa. The polyurethane was cured for 18 hours at 65° C. in an inert atmosphere, after which the stamps were removed from the surfaces of the substrates. Template bacteria were washed off with ethanol and PBS, leaving behind selective binding cavities on the surfaces of the substrates. Thus, surface-imprinted polymers (SIPs) were prepared to be selective for each of E. coli and S. aureus.
A flow cell having an inside diameter of 6 mm and a height of 4 mm, with a total interior volume of 110 μl, was made of acrylic (available under the trademark PERSPEX®, from Lucite International, of Lancashire, United Kingdom). The flow cell was coupled to a potentiostat, and was sealed with an O-ring. The contact area between the flow cell and the potentiostat system was 28 mm2. The SIP-coated substrates (described in Example 2) were mounted horizontally and pressed mechanically onto a copper block, which served as a heat sink. The temperature T1 of the copper block was actively controlled by a proportional-integral-derivative (PID) controller with control parameters P=1, I=8, and D=0, and measured by a thermocouple. The temperature T1 of the copper block was maintained at 37.00° C.
A second thermocouple was positioned above the surface of the SIP-coated substrates, which measured the temperature T2 in the liquid. The thermal resistance, abbreviated as Rth (° C./W), was determined by dividing the temperature difference (T1−T2) by the input power P (in Watts) consumed while keeping the temperature constant at 37.00° C. (Equation 1).
The SIP-coated substrates were stabilized in PBS buffer (pH=7.4) at the beginning of each experiment. Bacteria were introduced to the system by injecting 3 mL of a bacteria solution (1×107 CFU/mL in PBS) at a controlled flow rate of 2.5 mL/min. The SIP-coated substrates were stabilized, after which the SIP-coated substrates were flushed with PBS at a flow rate of 0.25 mL/min for 12 minutes (total volume 3 mL) to remove any unbound bacteria from the SIP layer. The HTM setup monitors the thermal resistance (Rth) at the solid-liquid interface at a rate of one measurement per second.
Microscopic imaging of the SIP-coated substrates was performed with a DM750 optical microscope, available from Leica Microsystems, of Diegem, Belgium. The SIP-coated substrates were imaged at magnifications 640× and 1000×. Software (ImageJ version 1.44p, available from National Institutes of Health, Bethesda, Md., USA) was used to determine the number of cell imprints per unit area on microscopic images of the SIP-coated substrates. The average surface coverage of cell imprints was calculated based on cell imprint counts of three different samples for each type of SIP-coated substrate and at five locations on each SIP-coated substrate.
Optical microscopic analysis of a SIP surface imprinted with E. coli (
A SIP-coated substrate was formed and imprinted with living E. coli cells in PBS (concentration 1×107 CFU/mL) as described in Examples 1 and 2. The SIP-coated substrate was mechanically pressed with its non-coated, polished backside onto a copper block, to ensure thermal contact between the SIP-coated substrate and the copper block. The SIP-coated substrate was placed in a flow cell, which was filled with PBS. The Rth signal of the SIP-coated substrate was allowed to stabilize for 60 minutes. Dead bacteria were introduced into the flow cell for 72 s at a flow rate of 2.5 mL/min. The flow was stopped, and the Rth signal was allowed to stabilize for 60 min, allowing the bacteria to sediment towards the SIP surface. Any unbound bacteria were removed by flushing the flow cell with PBS for 12 minutes at a rate of 0.25 mL/min. After a 60-minute stabilization interval, the experiment was repeated with living E. coli cells. The results of this experiment are shown in
The thermal resistance tests described in Example 5 and in
SIP-coated substrates were formed and imprinted with S. aureus cells (gram-positive bacteria) and E. coli cells (gram-negative bacteria) as described in Examples 1 and 2. The SIP-coated substrates were mechanically pressed with their non-coated, polished backsides onto copper blocks, to ensure thermal contact between the SIP-coated substrates and the copper blocks. The SIP-coated substrates were placed in a flow cell, which was filled with PBS. Time-dependent Rth data were acquired by consecutively exposing the SIP-coated substrates to analogue non-target bacteria and target bacteria. The flow cell was flushed at a controlled velocity between both exposure events.
Thus,
Portions of a stock solution of E. coli cells in PBS with a concentration of 1×107 CFU/mL were diluted 100, 50, 20 and 10 times, and a SIP-coated substrate (imprinted with E. coli, as described in Examples 1 and 2) was consecutively exposed to an increasing concentration of target E. coli cells in a flow cell. In between each exposure step, the flow cell was rinsed with ethanol for 12 minutes at a rate of 0.25 mL/min, followed by a rinse with PBS for 12 minutes at a rate of 0.25 mL/min. The results of this experiment are shown in
The thermal resistance increased when the E. coli cells were added, and the increases appear to be concentration-dependent. The time-dependent thermal resistance data shown in
The dose-response curve follows an empirical, exponential fit function according to the formula:
where c is the concentration of E. coli, and A, B, and C are constants. The exponential fit drawn through the obtained data in
The sensitivity tests described in Example 7 and
A solution was prepared containing both E. coli and S. aureus cells in a 1:99 ratio. The total concentration of bacteria was 1×107 CFU/mL. This mixture was used in a progressive enrichment experiment.
A SIP-coated substrate was imprinted with E. coli, as described in Example 2. The substrate was exposed three consecutive times to the mixture, and the substrate was flushed with buffer between each exposure event. The results are shown in
The saturation level at each step (indicated using the scale on the right of
Without being bound to any particular theory, it appears that both target and analogue cells bound to the SIP-coated substrate in the first exposure. After flushing, the signal fell back to a value that did not significantly differ from the baseline value. The total concentration of target cells (E. coli) in the mixture was only 1×105 CFU/mL, which is below the LoD determined in Example 7. Moreover, the E. coli cells were outnumbered 99:1 by S. aureus cells, an analogue bacteria that also bind to the microcavities in the SIP-coated substrate. E. coli cells cannot bind to microcavities that are already occupied by S. aureus cells. The analogue bacteria may also prevent the target bacteria from interacting with the SIP-coated substrate, due to steric hindrance.
These problems may be at least partially overcome by increasing the number of exposure cycles. With each cycle, the signal appeared to saturate and eventually reach the LoD, indicating that enrichment may improve the sensitivity of the SIP-coated substrate and may enable it to detect lower concentrations of bacteria in increasingly complex mixtures.
Seven bacteria-imprinted polyurethane layers selective to E. coli, S. aureus, K. pneumoniae, P. aeruginosa, S. epidermidis, A. baumannii, and E. coli K-12 were formed as described in Example 2. The polyurethane layers were placed on aluminum substrates in flow cells as described in Example 3. The flow cells were each configured to vary the temperature T1 of the copper block a function of time.
Each substrate was subjected to increasing concentrations of target bacteria in buffer solution. For each concentration of target bacteria, the temperature T1 was kept constant for a period of time, then varied to apply a thermal wave. The temperature under the substrate was kept constant at 37° C. by applying power P. The temperature T2 of the liquid flow cell was monitored in time. The thermal resistance (i.e., Rth=(T1−T2)/P) was also monitored over time. The results are shown in
These results show that the temperature (T2) in the liquid flow cell decreases when the amount of target bacteria in the flow cell increases. This appears to indicate that bacteria are binding to the polyurethane on the substrate, increasing the thermal resistance (Rth) at the solid-liquid interface, which in turn causes T2 to drop.
The thermal waves at each concentration were analyzed, and are shown in
The data in
It has been unexpectedly discovered that the methods and devices described herein may be used to discriminate not only between strains of similar bacteria, but also between living and dead bacteria of the same strain. Without being bound to any particular theory, it appears that the difference in surface chemistry between living and dead E. coli is sufficient to discriminate between them, despite their morphological similarities.
Furthermore, it has been unexpectedly discovered that rinsing non-target analytes (e.g., bacteria similar but not identical to a target analyte bacteria) can increase the detection capability of a polymer material by freeing binding sites of non-target analytes without removing target analytes from other binding sites. Thus, binding sites that were initially occupied by target analytes may remain filled, and binding sites that were initially occupied by non-target (but analogue) analytes may be cleared for re-binding with another analyte (in particular, with the target analyte). Analogue bacteria may bind to imprints to some extent, possibly due to the presence of bacteria-specific functional groups on the membrane of the cells that are compatible to some of the functional groups inside the imprints. However, the bond does not appear to withstand shear forces provided by flushing. The target bacteria, on the other hand, appear to remain firmly bound to the polymer, such that the thermal resistance remains at an elevated level even after flushing. Such clearing and re-binding may be useful for characterizing complex mixtures of similar or related analytes because related analytes may tend to weakly bind to sites imprinted for one another. By clearing and re-binding analytes, lower concentrations of the target analyte may be detected.
The methods and devices described herein may be used in conjunction with steady-state or thermal-wave analysis techniques. Various shapes of substrates may be used, and data (e.g., temperature) may be collected at various points, such as in the liquid to be analyzed, in a substrate coated with polymer material, or in a coated thermocouple.
Methods described herein may be used to provide real-time or nearly real-time characterization of bacteria that is conventionally performed in laboratories having complex equipment and highly trained personnel. Thus, the methods and devices may enable faster and cheaper data collection, and may enable improved outcomes by, for example, identifying bacterial outbreaks within a population. Such methods may be beneficial in health care, environmental and food safety (e.g., by detecting water-, air, and food-borne bacteria), and counter-terrorism (e.g., by detecting anthrax, etc.).
While the present disclosure has been described herein with respect to certain illustrated embodiments, those of ordinary skill in the art will recognize and appreciate that it is not so limited. Rather, many additions, deletions, and modifications to the illustrated embodiments may be made without departing from the scope of the disclosure as hereinafter claimed, including legal equivalents thereof. In addition, features from one embodiment may be combined with features of another embodiment while still being encompassed within the scope of the disclosure as contemplated by the inventors. Further, embodiments of the disclosure have utility with different and various detection devices and methods.
