Ultra-sensitive detection of analytes with allosteric transcription factor interfaced microcantilevers

Abstract

Systems and methods for detecting one or more analytes in a liquid sample are disclosed herein using a microcantilever coated with polynucleotides that comprise operator sequences specific to allosteric transcription factors are provided herein. Presence of analytes in the sample release the allosteric transcription factors causing a detectable change in the configuration of the microcantilever.

Description

ULTRA-SENSITIVE DETECTION OF ANALYTES WITH ALLOSTERICTRANSCRIPTION FACTOR INTERFACED MICROCANTILEVERSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 625.752 filed on January 26. 2024. The content of which is incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant number 2319427 awarded by the National Science Foundation. The government has certain rights in the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The content of the electronic sequence listing (70258102609.xml; Size: 17,085 bytes; and Date of Creation: January 27, 2025) is herein incorporated by reference in its entirety’.BACKGROUND

[0004] Reliable access to safe drinking water is a growing challenge, as studies estimate that 80% of the global population expenences high levels of threat to water security-. One area of focus around water security is contamination of potable water supplies by harmful chemicals including ions, such as lead, cadmium, and fluoride, and small molecules, such as benzene, perfluoroalkyls, and glyphosate, which are toxic and can contribute to a range of negative health consequences. An ability to detect harmful chemicals in water quickly and accurately is thus critical to achieving water security for all.SUMMARY

[0005] In an aspect, provided herein is a system for detecting one or more analytes in a liquid sample, the system comprising: a microcantilever; a plurality of polynucleotides coupled to at least a portion of the microcantilever, wherein each of the polynucleotides comprises one or more operator sequences; and one or more allosteric transcription factors, wherein each of the one or more allosteric transcription factors binds to at least one of the one or more operator sequences and at least one of the one or more analytes; wherein, when the one or more allosteric transcription factors bind to the one or more operator sequences, the microcantilever bends; and wherein, whenthe one or more analytes bind to the one or more allosteric transcription factors, the one or more allosteric transcription factors cannot bind to the one or more operator sequences.

[0006] Each of the polynucleotides may be double stranded DNA (dsDNA). The dsDNA may be thiolated.

[0007] The microcantilever may comprise a silicon-based material. The microcantilever may be at least partly coated in gold.

[0008] Each of the polynucleotides may comprise at least two operator sequences. The two operator sequences may be separated by a spacer sequence. The spacer sequence may be between about 2 nucleotides and about 30 nucleotides. The spacer sequence may be about 6 nucleotides.

[0009] Each polynucleotide may comprise a sequence having at least 90% identity to one of SEQ ID NOs: 1-12.

[0010] The microcantilever may have a maximum dimension of between about 100 pm and about 1.5 mm. The microcantilever may have a length of between about 450 pm to about 520 pm, a width of between about 80 pm to about 100 pm, and a thickness of betw een about 1 pm to about 1.5 pm.

[0011] The one or more allosteric transcription factors may comprise at least one of TetR and CadC. In embodiments, the one or more allosteric transcription factors comprise CadC, and the one or more analytes comprise at least one of Pb2+and Cd2+. The system may detect Pb2+in the liquid sample at a concentration of less than 10 parts per billion (ppb); and / or the Cd2+in the liquid sample at a concentration of less than 10 ppb. In embodiments, the one or more allosteric transcription factors comprise TetR. and the one or more analytes comprise anhydrotetracycline (aTc). The system may detect aTc at a concentration of less than 1 pM.

[0012] The microcantilever may be in a microfluidic chip.

[0013] The system may further comprise at least one additional microcantilever; a plurality of additional polynucleotides coupled to at least a portion of the at least one additional microcantilever, wherein each of the additional polynucleotides comprises one or more additional operator sequences; and one or more additional allosteric transcription factors, wherein each of the one or more additional allosteric transcription factors binds to at least one of the one or more additional operator sequences and at least one additional analyte; wherein, when the one or more additional allosteric transcription factors bind to the one or more additional operator sequences, the at least one additional microcantilever bends; wherein, when the additional analytes bind to the one or more additional allosteric transcription factors, the one or more additional allosteric transcription factors cannot bind to the one or more additional operator sequences; and wherein the additional operator sequences of the polynucleotides coupled to each additional microcantilever are different.

[0014] In another aspect, provided herein is a method for detecting one or more analytes in a liquid sample using any of the systems described herein.

[0015] In another aspect, provided herein is a method for detecting one or more analytes in a liquid sample, the method comprising: contacting an allosteric transcription factor interfaced microcantilever to the sample; and detecting a de-bending of the microcantilever; wherein the allosteric transcription factor interfaced microcantilever comprises: a plurality of polynucleotides coupled to at least a portion of the microcantilever, wherein each of the polynucleotides comprises one or more operator sequences; and one or more allosteric transcription factors bound to the one or more operator sequences; wherein, when the one or more analytes bind to the one or more allosteric transcription factors, the one or more allosteric transcription factors are released from the one or more operator sequences; and wherein detection of de-bending indicates the presence of the one or more analytes.

[0016] Detecting the de-bending may comprise utilizing atomic force microscopy or an electrical detection method. The contacting may be done for betw een about 15 minutes and about 30 minutes.

[0017] Each of the polynucleotides may be double stranded DNA (dsDNA). The dsDNA may be thiolated.

[0018] The microcantilever may comprise a silicon-based material. The microcantilever may be at least partly coated in gold.

[0019] Each of the polynucleotides may comprise at least two operator sequences. The two operator sequences may be separated by a spacer sequence. The spacer sequence may comprise between about 2 nucleotides to about 30 nucleotides. Each polynucleotide may comprise a sequence having at least 90% identity to one of SEQ ID NOs: 1-12.

[0020] The one or more allosteric transcription factors may comprise at least one of TetR and CadC. In embodiments, the one or more allosteric transcription factors comprise CadC, and the one or more analytes comprise at least one of Pb2+and Cd2+. The Pb2+in the sample may be detected at a concentration of less than 10 parts per billion (ppb); and / or the Cd2+in the liquid sample may be detected at a concentration of less than 10 ppb. In embodiments, the one or more allosteric transcription factors comprise TetR, and the one or more analytes comprise anhy dr otetracy cline (aTc). aTc in the sample may be detected at a concentration of less than 1 pM.

[0021] The microcantilever may have a maximum dimension of between about 100 pm and about 1.5 mm. The microcantilever may have a length of between about 450 pm to about 520 pm, a width of between about 80 pm to about 100 pm, and a thickness of between about 1 pm to about 1.5 pm.

[0022] The allosteric transcription factor interfaced microcantilever may be in a microfluidic chip.

[0023] The liquid sample comprises water or a beverage.The method may further comprise: contacting at least one additional allosteric transcription factor interfaced microcantilever to the sample; and detecting a de-bending of the additional microcantilever; wherein each of the additional allosteric transcription factor interfaced microcantilevers comprises: a plurality' of additional polynucleotides coupled to at least a portion of the additional microcantilever, wherein each of the additional polynucleotides comprises one or more additional operator sequences; and one or more additional allosteric transcription factors bound to the one or more additional operator sequences; wherein, when one or more additional analytes bind to the one or more additional allosteric transcription factors, the one or more allosteric transcription factors are released from the one or more additional operator sequences; wherein detection of de-bending indicates the presence of the one or more additional analytes; and wherein the additional operator sequences of the polynucleotides coupled to each additional microcantilever are different.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIGS. 1A-1D. Interfacing microcantilevers with allosteric transcription factor biosensors. (A) Microcantilevers are first coated with double stranded DNA (dsDNA) containing aTF operator sequences. aTF binding to the immobilized dsDNA causes cantilever bending. Binding of a specific chemical ligand to the aTF causes an allosteric change, releasing aTFs from the dsDNA, resulting in cantilever de-bending which can be detected optically. (B) Microcantilever bending in response to various concentrations of TetR. (C) Microcantilever de-bending with 1 pM TetR and various concentrations of aTc. Carbenicillin (Carb.), an antibiotic not recognized by TetR, was used as a control. De-bending is computed as the difference in microcantilever bending (deflection) between the sensing microcantilever, induced with aTc, and the average bending of the control microcantilevers, induced with 1 pM carbenicillin (Methods. FIG. 3). (D) A dose response curve for de-bending at 11 minutes. Data in B-D are for n = 3 technical replicates, where each technical replicate is represented by a solid line (B, C) or point (D). Shaded regions in B, C represent ± one standard deviation about the mean of all technical replicates at each condition. Full data plots can be found in FIG. 4.

[0025] FIGS. 2A-2B. Microfluidic chip design and microcantilever design. An SEM image of the microfluidic chip design (A) and microcantilever (B) used for microcantilever bending and de- bending experiments. The microcantilever is 508 pm long (base to tip) by 95.3 pm wide. The microfluidic chips, essential components in microscale experiments, were intricately fabricated through photolithography and etching by Nanoink Inc in 2006.

[0026] FIGS. 3A-3C. Schematic depiction of microcantilever bending and de-bending measurements. (A) Microcantilever deflection is measured as the distance of the tip of the microcantilever from the origin of the undeflected cantilever. (B) aTF-DNA interactions cause microcantilever bending, where the distance the cantilever bends is equivalent to the measured deflection. (C) Upon the addition of a ligand, aTF-ligand interactions cause an allosteric change, resulting in unbinding of the aTF and a de-bending of the cantilever (‘‘induced,’' left). The addition of a control compound that does not bind the aTF does not result in an allosteric change or microcantilever de-bending. The difference in microcantilever deflection between the ligand and control conditions is used to calculate ligand-induced microcantilever de-bending.

[0027] FIG. 4. Raw AFM microcantilever deflection data for studies involving the anhydrotetracycline-TetR system. Left column: Bending in the presence of varying concentrations of TetR. Right column: De-bending in the presence of either tetracycline (“Tet”) or carbenicillin (‘’Carb). Each plot is labeled with the DNA design used. For example, the DNA design “2X3BP” indicates two tetO operator sites per dsDNA with three base pair spacing between them, and the DNA design “IX” indicates one operator site for dsDNA. A list of all DNA designs used is in Table 1. In the plot legends, aTc indicates anhydrotetracycline. Carb, indicates carbenicillin. Chlor, indicates chloramphenicol, and Kan. indicates kanamycin. Each solid line displayed in the plots corresponds to a single microcantilever's deflection over time as measured via AFM (one cantilever = one technical replicate), while the shaded region represents plus / minus one standard deviation about the mean of all technical replicates at each condition. For plots with no shaded region, only one cantilever was studied, and therefore standard deviation could not be computed. For de- bending experiments, 10 pL of 1 pM TetR solution was used to bend the microcantilevers, followed by the addition of 10 pL of the indicated analyte solution (see Materials and Methods).

[0028] FIGS. 5A-5E. Varying the number and spacing of DNA operator sequences can tune microcantilever detection performance. (A) DNA sequences were modified to contain multiple tetO operator sequences with different spacings between them. The notation NXMBP indicates N operator sequences were included with a spacing of M base pairs. The example shown in the schematic is 2X6BP tetO DNA design. (B) Bending data collected at 11 minutes with different DNA designs. DNA sequences were modified to contain multiple TetR operator sites with different spacings between them. The notation NXMBP indicates N operator sites were included with a spacing of M bps. Solid lines pass through the mean of the data points for each DNA design. The IX DNA design contains three replicates for each TetR condition, while the other DNA designs contain one replicate for each TetR condition. Time course data plots can be found in FIG. 4. (C) Observed de-bending data collected at 11 minutes using 1 pM TetR and a range ofanhydrotetracycline (aTc) concentrations with different DNA designs. (D) De-bending at 11 minutes using the 2X6BP tetO DNA design with 1 pM TetR and a range of aTc concentrations after leaving the DNA-coated microcantilevers at room temperature for 0 days (circles), 15 days (squares), or 30 days (triangles). Each line passes through the mean of the data points for a given day. (E) Observed de-bending at 11 minutes using the 2X6BP tetO DNA design with 1 pM TetR and inducing with different antibiotics including anhydrotetracycline (aTc), chloramphenicol (Chlor.) and kanamycin (Kan.). De-bending was calculated using a 1 pM carbenicillin control condition as in FIG. 1. Individual data points in (C-E) represent technical replicates, each corresponding to a single microcantilever. Solid lines in (C) and (D) pass through the mean of the data points, while the error bars in (D) correspond to + / - one standard deviation about the mean. For the 2X6BP tetO DNA case in panel (C). the ligand concentration at which the bending magnitude was distinguishable from the carbenicillin induction control were determined using a two-tailed, homoscedastic Student’s t-test against the carbenicillin control condition, and their P value ranges are indicated with asterisks (***P < 0.001, **P = 0.001-0.01, *P = 0.01-0.05). Time course data plots can be found in FIG. 4.

[0029] FIG. 6. Raw AFM microcantilever deflection data for studies involving the Cd-CadC system using the 2X6BP DNA design. Each solid line displayed in the plots corresponds to a single microcantilever's bending or de-bending over time as measured via AFM (one cantilever = one technical replicate), while the shaded region represents plus / minus one standard deviation about the mean of all technical replicates at each condition. For plots with no shaded region, only one cantilever was studied, and therefore standard deviation could not be computed. In the plot legends, “Carb.” indicates carbenicillin. For de-bending experiments, 10 pL of 5 pM CadC was used to bend the cantilever, followed by the addition of 10 pL of the indicated concentration of analyte solution (see Methods).

[0030] FIG. 7. Raw AFM microcantilever deflection data for studies involving the Pb-CadC system using the 2X6BP DNA design. A list of all DNA designs used can be found in Table 1. In the plot legends, Carb, indicates carbenicillin. Each solid line displayed in the plots corresponds to a single microcantilever's deflection over time as measured via AFM (one cantilever = one technical replicate), while the shaded region represents plus / minus one standard deviation about the mean of all technical replicates at each condition. For plots with no shaded region, only one cantilever was studied, and therefore standard deviation could not be computed. For de-bending experiments, 10 pL of 5 pM CadC was used to bend the cantilever, followed by the addition of 10 pL of the indicated concentration of analyte solution (see Methods). For studies involving drinking water, the concentration of Pb was determined via FAAS. Importantly, the unspiked drinking watercontained 0.25 ppb (1.2 nM) Pb.

[0031] FIGS. 8A-8E. Microcantilevers interfaced with the CadC transcription factor can detect nanomolar concentrations of Pb2+and Cd2+. (A) Schematic depicting the lead-CadC and cadmium- CadC sensing systems with the 2X6BP cadO DNA design and 5 pM CadC. (B) De-bending at 1 1 minutes for a range of PbCh concentrations. (C) De-bending at 11 minutes for a range of CdCh concentrations. (D) De-bending at 11 minutes for a range of PbCh concentrations after leaving the DNA-coated microcantilevers at room temperature for 0 days (circles). 15 days (squares), and 30 days (triangles). Each line passes through the mean of the data points for a given day. (E) De- bending at 11 minutes for different metals including Pb2+(PbCh), Zn2+(ZnSO-i) and Cu2+(CuCh). De-bending is computed as the difference in bending between the sensing microcantilever, induced with lead or cadmium, and the mean of the control microcantilevers, induced with 1 pM carbenicillin as in FIG. 1. Data shown are for n = 3 technical replicates, where each technical replicate, corresponding to a single microcantilever, is represented by a point. Each solid line passes through the mean of the data points. The ligand concentrations at which the bending magnitude was distinguishable from the carbenicillin induction control were determined using a two-tailed, homoscedastic Student’s t-test against the carbenicillin control condition, and their P value ranges are indicated with asterisks for the 2X6BP case (***P < 0.001, **P = 0.001-0.01, *P = 0.01-0.05). Time course data plots can be found in FIGS. 6, 7.

[0032] FIG. 9. Double equilibrium model of transcription factor binding to DNA and target compound. The double equilibrium model considers association / dissociation of anhydrotetracycline-TetR (top) and of TetR-7’e / G (bottom).

[0033] FIG. 10. ROSALIND double equilibrium analysis. Model prediction of fraction of DNA without TetR bound using the DNA template and TetR concentration studied in [3] with literature values for the association constants of anhydrotetracycline-TetR and TetR- / e / (9 The values used here are: KD,L = 7.94e-13 M, KD,i = 1.79e-10 M, [DNA]0= 25 nM, [TetR]0= 1250 nM, [aTC]0= 0.1 — 100 pM. Replotted induction curve (normalized) of the ROSALIND reaction from [3] using [DNA]0= 25 nM, [TetR]0= 1250 nM. Refer to [3] for methodological details ofthe ROSALIND experiment.

[0034] FIG. 11. Base case microcantilever double equilibrium analysis. A plot displaying the fraction of DNA without TetR bound as predicted by the model (circles) and an overlay of the normalized microcantilever de-bending data (squares) for the different DNA designs (see FIG. 5). The values used here are: KD L = 7.94e-13 M. KD i = 1.79e-10 M, [DNA]0= 4.15 — 4150[TetR]0= 500 nM, [aTC]0= 0.001 — 100 pM. Two different values of [DNA]0exploredcorrespond to ten times the maximum feasible concentration (F = 10) and one one-hundredth the maximum feasible concentration (F = 0.01), where the maximum feasible concentration is based on the estimated area that a dsDNA molecule would occupy on the microcantilever surface. There is negligible difference in the model’s prediction for the F = 10 and F = 0.01 cases and so those points overlap.

[0035] FIG. 12. Changing TetR concentration and the TetR-DNA association constant generates closer agreement between the double equilibrium model and observed de-bending data. A plot displaying the normalized microcantilever de-bending data (squares) for the different DNA designs along with an overlay of the fraction of DNA without TetR bound as predicted by the model (circles) using a lower initial TetR concentration (by two orders of magnitude) and a lower TetR- tetO dissociation constant (by -four orders of magnitude). The values used here are: KD,L = 7.94e- 13 M, KD,I — 2.27e-14 M, [DNA]0= 415 0.05 - 50 pM so totalaTC concentration ranges from 0.1 - 100 pM.

[0036] FIG. 13. DNA coating concentration affects maximum achievable microcantilever bending. A plot displaying how microcantilever bending is affected by the concentration of dsDNA used to coat the microcantilevers. After coating, bending was performed using 10 pL of 1 pM TetR solution. The DNA used for this study is the “ IX” tetO DNA design, meaning one TetR operator sequence per dsDNA. Data are for n = 3 technical replicates, where each technical replicate is represented by a solid line. Shaded regions represent plus / minus one standard deviation about the mean of all technical replicates at each condition.

[0037] FIGS. 14A-14D. Microcantilevers interfaced with the CadC transcription factor can detect Pb2+in drinking water. (A) Drinking water was spiked with varying amounts of PbCb (0, 0.01, 0.05, and 0.1 pM) and de-bending was measured using the 2X6BP cctdO DNA design and 5 pM CadC (gray points). The concentration of Pb in these spiked samples was measured via GFAAS. These results are presented with the previous study wherein PbCb was added to 18 MOhm water (purple points, data from FIG. 8B). (B) A best fit line was computed to correlate loglO([Pb] pM) and de-bending magnitude on the range 0.001 - 0.1 pM Pb (R2=0.9773) for the study performed in 18 MOhm water (purple points). (C) The best fit line from (B) correlates well with the de- bending measured for the spiked drinking water samples (R2=0.8465). (D) The correlation generated in (B) was used to predict the concentration of Pb in the spiked drinking water samples based on the de-bending magnitudes measured for those samples. These predictions (y-axis) were plotted against the actual concentration of Pb as measured via GFAAS (x-axis) along with the line y=x for ease of viewing. De-bending is computed as the difference in bending between the sensingmicrocantilever, induced with Pb, and the mean of the control microcantilevers, induced with 1 pM carbenicillin in 18 MOhm water as in FIG. 1. Data show n are for n = 3 technical replicates, where each technical replicate, corresponding to a single microcantilever, is represented by a point. Each solid line passes through the mean of the data points. Time course data plots can be found in FIG. 7.

[0038] FIG. 15. Calibration plot for lead measurement by HR-CS-GFAAS. Error bars representing one standard deviation about the mean of each point are not visible because of their small magnitudes. The calibration slope (a) is provided in the plot legend with its uncertainty computed using the value of a Student’s t test for a continuous random variable (t), the residual sum of squares using the best fit slope (RSS), degrees of freedom (m), and sample corrected sum of squares (Sxx) as outlined Equation 1 (FIG. 16).

[0039] FIG. 16. Equation 1.DETAILED DESCRIPTION

[0040] The present disclosure relates to systems and methods for detecting analytes in a liquid. In various aspects, as discussed herein, an allosteric transcription factor (aTF) interfaced cantilever can be utilized to provide ultra-sensitive detection of the analytes. As discussed herein, the microcantilevers can be functionalized for analyte detection by patterning the microcantilever surface with DNA containing aTF operators. Binding of the aTF to the DNA causes microcantilever bending, while the presence of a specific chemical that binds to the aTF releases the aTF from the operator and causes microcantilever de-bending. By detecting the bending and / or de-bending of the microcantilever, the present system and methods can detect various analytes at low concentrations.

[0041] The systems and methods disclosed herein can be used for various applications, such as water uality monitoring, human health diagnostics, food quality monitoring, environmental monitoring, deployment in continuous monitoring devices, use within ‘smart’ cities applications to do real-time communication of monitoring targets, and use as components of deploy able devices for monitoring or in consumer electronics.

[0042] The systems and methods described herein provide numerous advantages. They improve upon the sensitivity of cell-free biosensors used in other formats. They can create a digital read out that can be stored, processed, logged and communicated. They create a fast response which can be made faster through signal processing. They're amenable to continuous monitoring schemes by embedding within a device chamber.

[0043] Accordingly, in a first aspect, a system for detecting one or more analytes in a liquidsample, is provided herein, the system comprising: a microcantilever; a plurality of polynucleotides coupled to at least a portion of the microcantilever, wherein each of the polynucleotides comprises one or more operator sequences; and one or more allosteric transcription factors, wherein each of the one or more allosteric transcription factors binds to at least one of the one or more operator sequences and at least one of the one or more analytes; wherein, when the one or more allosteric transcription factors bind to the one or more operator sequences, the microcantilever bends; and wherein, when the one or more analytes bind to the one or more allosteric transcription factors, the one or more allosteric transcription factors cannot bind to the one or more operator sequences.

[0044] The term “microcantilevef’ is a micrometer sized device comprising a beam that is anchored only at one end. A microcantilever physically bends when subjected to stresses induced by molecular binding events on its surface. As shown in the examples, the binding of proteins to polynucleotides coated onto the microcantilevers disclosed herein is sufficient to measurably bend the microcantilever from its original configuration, such as depicted in FIG. 1A. The microcantilever is pliable, such that when the proteins are released or become unbound from the microcantilever, the microcantilever can de-bend back toward its original configuration. In embodiments, the microcantilever is fabricated using silicon-based materials. Silicon on Insulator (SOI) waiters may be used to define the cantilever geometry. This approach leverages well- established silicon-based micromechanical technology' to achieve precise control over dimensions and mechanical properties, ensuring high reproducibility and uniformity. The microcantilever may comprise or consist of a silicon-based material. The microcantilever may further comprise a gold- coating. The microcantilever may be wholly or partially coated in gold.

[0045] For the readout mechanism, MOSFET devices (which uses silicon based technology) may be incorporated directly on the chip. This integration allows for efficient, scalable, and low-cost electronic signal processing, making the system highly suitable for large-scale manufacturing and deployment.

[0046] The microcantilever may have a maximum dimension of 100 pm or more, or 1.5 mm or less. The maximum dimension may be between about 100 pm and about 1.5 mm, or any size or range in between. The maximum dimension may be between about 100 pm and about 600 pm. As used herein "maximum dimension” refers to the longest linear dimension of the microcantilever taken along the major axis. A maximum dimension is the greatest value of a length, height, or thickness of the microcantilever. In one example aspect, a microcantilever can have the dimensions of 500 x 100 x 1 microns, and would exhibit a maximum dimension of 500 microns. In embodiments, the microcantilever has a length of between about 450 pm to about 520 pm, a width of between about 80 pm to about 100 pm, and a thickness of between about 1 pm to about 1.5 pm.

[0047] The microcantilever may be functional in at least about 1 qL of liquid. The microcantilever may be functional in no more than about 50 qL of liquid. The microcantilever may have a volume requirement of between about 1 qL and about 50 qL or any volume or range in between. In embodiments, the volume requirement is between about 5 qL and about 25 qL.

[0048] The polynucleotides may be coupled to the microcantilever in any manner. In exemplary embodiments, the polynucleotides include a thiolated 5‘ end, which can covalently attach to a gold surface or other surface of a microcantilever.

[0049] Any number of polynucleotides can be coupled to a microcantilever, and such number may vary depending upon the length of the polynucleotide strands, their conformation, and / or the surface functionalization techniques used on the microcantilever. The density of the polynucleotides on a microcantilever can be in a range of 1 x 1012 / cm2to 30 x 1013 / cm2, or 5.2 x 1012 / cm2to 9.8 x 1013 / cm2. In embodiments, the microcantilever has the dimensions of 500 x 100 x 1 microns and the density of the polynucleotides on the microcantilever is in a range of 1 x 1012 / cm2to 30 x 1013 / cm2, or 5.2 x 1012 / cm2to 9.8 x 1013 / cm2.

[0050] The terms '’polynucleotide." “polynucleotide sequence,” “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of genomic, natural, or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand).

[0051] The terms “nucleic acid” and “oligonucleotide,” as used herein, may refer to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D- ribose), and to any other type of polynucleotide that is an N glycoside of a purine or pyrimidine base. There is no intended distinction in length between the terms “nucleic acid”, “oligonucleotide” and “polynucleotide”, and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA. For use in the present methods, an oligonucleotide also can comprise nucleotide analogs in which the base, sugar, or phosphate backbone is modified as well as non-purine or non-pyrimidine nucleotide analogs. In exemplary embodiments, the polynucleotides comprise double-stranded DNA (dsDNA).

[0052] A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of tw o or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques known in the art. The term recombinant includes nucleic acids that have been alteredsolely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.

[0053] The polynucleotides may comprise one or more aTF operator sequences. The polynucleotides may comprise at least two, at least three, or at least four TF operator sequences. In such embodiments, the polynucleotides may comprise a spacer sequence that separate the aTF operator sequences. The spacer sequence can be any sequence and / or length. The spacer sequence may be tailored to modulate sensitivity and / or selectivity of the system and methods disclosed herein. The spacer sequence may be from about 2 nucleotides to about 30 nucleotides and any number of nucleotides or range in between. For example, the spacer sequence may be between about 2 nucleotides and about 20 nucleotides, between about 2 nucleotides and about 15 nucleotides, between about 2 nucleotides and about 10 nucleotides, or between about 3 and about 6 nucleotides. In exemplary7embodiments, the spacer sequence is about 6 nucleotides. The spacer sequence can include any nucleotides. In embodiments, the spacer sequence includes or consists of the nucleotides A or G. In exemplary embodiments, the spacer sequence includes the sequence GGA and / or one or more repeats of GGA.

[0054] The polynucleotides can include any aTF operator sequence. In embodiments, the polynucleotide comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to one or more of SEQ ID NOs: 1- 12. The polynucleotide can consist essentially of, or consist of, a polynucleotide sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to one or more of SEQ ID NOs: 1-12. The polynucleotide sequence may be one or more of SEQ ID NOs: 1-12.

[0055] An "operator ’. "operator sequence”, or "allosteric transcription factor operator sequence” is a sequence that serves as a binding site for regulatory proteins to control gene expression. As used herein, the operator sequence is a binding site for allosteric binding proteins. As used herein, “an allosteric transcription factor (aTF)” refers to transcription factors which, upon binding a small molecule, such as a ligand, undergo an allosteric change. The term “transcription factor” refers to a protein that regulates transcription of another gene, typically by interacting with one or more cisacting DNA sequences, e.g., an operator, in or near the promoter for the gene. A transcription factor may increase expression or decrease expression depending upon whether the transcription factor is activated or deactivated. A transcription factor may become activated or deactivated by an interaction with another molecule (e.g. , a ligand as described herein). Such transcription factorsare termed allosteric transcription factors (aTFs).

[0056] An allosteric transcription factor (aTF), as described herein has a site that binds an operator sequence and a separate site that binds a ligand / analyte. Upon binding of the analyte the aTF undergoes a conformational change that releases it from the operator sequence. Any aTF that can bind or unbind an operator sequence upon binding of a ligand and / or analyte of interest is suitable for use in the present system and methods. A non-limiting list of aTFs includes, but is not limited to, TetR. a CadC, OtrR, CtcS. MphR. MobR. QacR, TtgR. HucR. SmtB, CsoR. and AdcR. In a non-limiting aspect, the aTF can include a polypeptide sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO: 13 and / or 14.

[0057] Each allosteric binding protein may be specific to an operator sequence. In an exemplary embodiment, the aTF is TetR, and the operator sequence is a tetO sequence. Binding of anhydrotetracycline (aTc) to TetR releases it from the tetO operator sequence. In another exemplary7embodiment, the aTF is CadC, and the operator sequence is a cadO sequence. Binding of lead (Pb2+) and cadmium (Cd2+) to CadC release it from the cadO operator sequence.

[0058] When the aTF comprises CadC, the system described herein can detect Pb2+in a water or other liquid sample at a concentration of less than about 10 parts per billion (ppb), less than about 8 ppb, less than about 5 ppb, or as low as about 2 ppb. When the aTF comprises CadC, the system can detect Cd2+in a water or other liquid sample at a concentration of less than about 10 ppb, less than about 8 ppb, less than about 5 ppb. or as low as about 1 ppb. When the aTF comprises TetR the systems described herein can detect anhydrotetracycline in water or liquid sample at a concentration of less than about 1 micromolar, less than about 0.5 micromolar, less than about 0. 1 micromolar, or as low as about 0.01 micromolar. The liquid sample may be water, a beverage, or any other liquid.

[0059] The aTF interfaced microcantilevers can be used as a biosensing system to detect a variety of materials. A non-limiting list of example ligands can include a metal ion, an organic compound, an inorganic compound, etc. Such detectable materials include but are not limited to water contaminants such as lead, cadmium, and fluoride, and small molecules, such as benzene, perfluoroalkyls, and glyphosate. Detectable materials include, but are not limited to, antibiotics, such as tetracycline, doxycycline, oxytetracycline, chlortetracycline, erythromycin, azithromycin, clarithromycin, and / or roxithromycin; small molecules, such as benzoic acid, benzalkonium chloride, naringenin, and / or uric acid; and / or metals and / or metalloids, such as zinc, copper, lead, arsenic, and / or cadmium.

[0060] The systems may comprise additional microcantilevers that each comprise differentpolynucleotides that have different operator sequences. Each microcantilever in a system may thus be configured to detect a different analyte or a different set of analytes. The microcantilevers may be assembled in a microfluidic device.

[0061] A “microfluidic device” or “microfluidic chip” may include fluidically interconnected circuit elements, including but not limited to region(s), flow path(s), channel(s), chamber(s), and / or pen(s), and at least one port configured to allow the fluid (and, optionally, micro-objects suspended in the fluid) to flow into and / or out of the microfluidic device. Typically, a microfluidic circuit of a microfluidic device will include a flow region, which may include a microfluidic channel, and at least one chamber, and will hold a volume of fluid of less than about 1 mL, e.g., less than about 750, 500. 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 pL. The microfluidic circuit may hold about 1-2, 1-3, 1-4. 1-5, 2-5, 2-8, 2-10, 2-12, 2-15, 2-20, 5-20, 5-30, 5-40, 5-50, 10-50, 10-75, 10-100, 20-100, 20-150, 20-200, 50-200, 50-250, or 50-300 pL. The microfluidic circuit may be configured to have a first end fluidically connected with a first port (e.g., an inlet) in the microfluidic device and a second end fluidically connected with a second port (e.g., an outlet) in the microfluidic device.

[0062] The system may be coupled to a device that provides a digital read out of the bending and de-bending measurements.

[0063] The terms “percent identity" and “% identity” refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity for a nucleic acid sequence may be determined as understood in the art. (See, e.g., U.S. Patent No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCB1) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastn.” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST 2 Sequences” tool can be used for both blastn and blastp.

[0064] Regarding polynucleotide sequences, percent identity may be measured over the length of an entire defined polynucleotide sequence, for example, as defined by a particular SEQ ID number,or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

[0065] As used herein, the terms “peptide,” “polypeptide,” and “protein,” refer to molecules comprising a chain a polymer of amino acid residues joined by amide linkages. The term “amino acid residue,” includes but is not limited to amino acid residues contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H). isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Vai or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acid residue” also may include nonstandard or unnatural amino acids. The term “amino acid residue” may include alpha-, beta-, gamma-, and delta-amino acids.

[0066] In some embodiments, the term “amino acid residue” may include nonstandard or unnatural amino acid residues contained in the group consisting of homocysteine, 2- Aminoadipic acid, N- Ethylasparagine, 3-Aminoadipic acid. Hydroxy lysine, [3-alanine. -Amino-propionic acid, allo- Hydroxylysine acid, 2-Aminobutyric acid, 3 -Hydroxy proline, 4-Aminobutyric acid, 4- Hydroxyproline, piperidinic acid, 6-Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2-Aminoisobutyric acid, N-Methylglycine, sarcosine, 3-Aminoisobutyric acid, N- Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4-Diaminobutyric acid, N- Methylvaline, Desmosine, Norvaline, 2,2'-Diaminopimelic acid. Norleucine, 2,3- Diaminopropionic acid. Ornithine, and N -Ethylglycine. The term “amino acid residue” may include L isomers or D isomers of any of the aforementioned amino acids.

[0067] Other examples of nonstandard or unnatural amino acids include, but are not limited, to a p-acetyl-L-phenylalanine, a p-iodo-L-phenylalanine, an O-methyl-L-tyrosine, a p- propargyloxyphenylalanine, ap-propargyl-phenylalanine. an L-3-(2-naphthyl)alanine, a 3-methyl- phenyl alanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAcp -serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-bromophenylalanine, a p-amino-L-phenylalanine, an isopropyl-L- phenylalanine, an unnatural analogue of a tyrosine amino acid; an unnatural analogue of aglutamine amino acid; an unnatural analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an unnatural analogue of a methionine amino acid; an unnatural analogue of a leucine amino acid; an unnatural analogue of a isoleucine amino acid; an alky l, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, ufahor, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or a combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; a metal binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photocaged and / or photoisomerizable amino acid; a biotin or biotinanalogue containing amino acid; a keto containing amino acid; an amino acid comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid; a carbon-linked sugar-containing amino acid; a redox-active amino acid; an a-hydroxy containing acid; an amino thio acid; an a, a disubstituted amino acid; a [3-amino acid; a y-amino acid, a cyclic amino acid other than proline or histidine, and an aromatic amino acid other than phenylalanine, tyrosine or try ptophan.

[0068] In a second aspect, a kit is provided herein, the kit comprising any of the microcantilevers described herein, wherein the microcantilevers comprise a plurality' of polynucleotides each comprising one or more operator sequences; and one or more allosteric transcription factors; wherein the microcantilevers and allosteric transcription factors are provided in separate packaging.

[0069] In a third aspect, provided herein is a method for detecting one or more analytes in a liquid sample, the method comprising: contacting an allosteric transcription factor interfaced microcantilever to the sample; and detecting a de-bending of the microcantilever; wherein the allosteric transcription factor interfaced microcantilever comprises: a plurality of polynucleotides coupled to at least a portion of the microcantilever, wherein each of the polynucleotides comprises one or more operator sequences; asnd one or more allosteric transcription factors bound to the one or more operator sequences; wherein, when the one or more analytes bind to the one or more allosteric transcription factors, the one or more allosteric transcription factors are released from the one or more operator sequences; and wherein detection of de-bending indicates the presence of the one or more analytes.

[0070] An allosteric transcription factor interfaced microcantilever is a microcantilever coated in the polynucleotides, wherein the polynucleotides are bound by the aTFs. The microcantilever, aTFs, and polynucleotides may be any of those described herein.

[0071] Atomic force microscopy may be utilized to detect the bending and de-bending of the microcantilever. Alternatively, one or more metal-oxide-semiconductor field-effect transistors (MOSFETs) embedded in the microcantilevers may be utilized as a mechanism for electrical detection of the bending and de-bending of the microcantilever.

[0072] The liquid sample may be water, a beverage, or any other liquid. The methods may provide an indication of the presence of an analyte or ligand in a liquid sample within 30 minutes, w ithin 20 minutes, or within 15 minutes from the time of exposing the sample to the microcantilever.

[0073] The systems and methods disclosed herein can include multiplexed arrays of microcantilevers, which can detect multiple analytes at the same time or at about the same time. For instance, in embodiments, the systems and methods disclosed herein can include a first allosteric transcription factor interfaced microcantilever that detects a first analyte and a second allosteric transcription factor interfaced microcantilever that detects a second analyte.

[0074] Miscellaneous

[0075] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”

[0076] As used herein, “about”, “approximately,” “substantially.” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term w hich are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0077] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter. Embodiments recited as “including.” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements.

[0078] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in thesame, the modal verb “may’’ refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb ‘'may” has the same meaning and connotation as the auxiliary verb “can.”

[0079] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0080] In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g.. “a system having at least one of A. B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0081] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between anydefinitions and / or description found in the cited references.

[0082] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherw ise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0083] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carry ing out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0084] EXAMPLES

[0085] Example 1 - Rapid, ultra-sensitive detection of water contaminants with aTF interfaced microcantilevers

[0086] Introduction

[0087] Recent advances in synthetic biology7have created cell-free biosensors that can detect harmful chemicals in water quickly and accurately, offering an alternative to laboratory-based analytical chemistry approaches that are costly and slow [3-6], Cell-free biosensors work by repurposing allosteric transcription factors (aTFs) within cell-free gene expression reactions to sense chemicals in water [3. 7-10], For apo-repressor aTFs, reporter templates are configured to contain an aTF operator sequence in front of the reporter gene: in the absence of the target chemical the aTF binds to the operator and blocks expression of the reporter; when the target chemical is present, it binds to the aTF causing it to unbind from the operator and allow gene expression to continue [3], In this way. cell-free biosensors exploit the binding / unbinding of aTFs to DNA in the absence / presence of target chemicals to synthesize a reporter gene, which typically produces either a fluorescent [3] or colorimetric read out that can be detected by visual inspection.

[0088] Cell-free biosensing reactions can be assembled from components, lyophilized, and then used by simply adding a water sample and waiting to observe an output signal [3], By changing the aTF, they can be configured to detect a range of compounds including tetracycline [3,7], Hg2+[7], Cd2+[3], Pb2+[3], azithromycin [3]. and benzalkonium chloride [3], among others [3], Cell- free biosensors are also cost effective (<$1 USD / unit) [3, 6], and can be manufactured and distributed for use in the field at the point-of-need by non-expert users [3, 6, 11], making them excellent candidates for increasing our collective capacity to scale water quality measurements to what is needed to address global challenges.

[0089] While promising, cell-free biosensors are still under development and currently have two key limitations. The first is with respect to limits of detection (LOD). Biosensor LODs are often determined by the biosensor system used. For some contaminants such as fluoride, cell-free biosensors can achieve LODs that are relevant to Environmental Protection Agency (EP A) or World Health Organization (WHO) action limits [6], while for others such as cadmium, cell-free biosensors have not been able to meet action limits [3], The second is read out quantitation. Most cell-free biosensors are configured to produce optical signals, making quantifying their signal difficult without sophisticated equipment not amenable to field deployment. There is thus great potential in interfacing cell free biosensor reactions with digital read out platforms that can facilitate broad deployment, as recognized by recent work

[0012] ,

[0090] The systems and methods disclosed herein address these limitations by creating an interface between cell-free biosensors and a highly sensitive nano-mechanical sensing platform that can be utilized to detect the fundamental binding / unbinding reactions that occur during aTF-mediated chemical detection. Specifically, we leverage microcantilevers - micrometer sized devices that physically bend when subjected to stresses induced by molecular binding events on their surfaces [13-15], When coated with binding interaction partners, they can be used to detect important molecules. For example, microcantilevers have been coated with antibodies, enabling them to detect a range of molecules through binding such as biotin

[0015] , goat anti rabbit IgG

[0015] , and SARS-CoV-2 spike protein

[0016] , with low limits of detection (LODs) (as low as hundreds of fM

[0016] ). In addition to low LODs, microcantilevers have a number of advantages including their small size (-500 x 95 x 1 pm

[0016] ), ability to parallelize in a detection array [17, 18], low sample volume requirements (as low as 4 pL

[0014] ), and their ability to provide a continuous digital read out

[0015] , When coupled with metal-oxi de-semiconductor field-effect transistor (MOSFET) technology, microcantilevers are even compatible with direct monolithic integration for integrated circuits, enabling their deployment in a wide range of electronic devices

[0015] ,

[0091] To address current limitations of cell-free biosensors and expand the capabilities of nanomechanical sensors, a new approach was created to interface the RNA Output Sensors Activated by Ligand INDuction (ROSALIND) [3] aTF biosensing system with microcantilevers by coating the microcantilever surface with double-stranded DNA (dsDNA) containing aTF operatorsequences (FIG. 1). In this way, binding of the aTF to the DNA causes microcantilever bending, while the presence of a specific chemical that releases the aTF from the DNA causes microcantilever de-bending. We first established the system using optical detection of microcantilever deflection and DNA encoding the operator sequence for the TetR aTF which senses tetracycline antibiotics including anhydrotetracycline (aTc). After exploring the effects of incorporating multiple aTF operators within the bound DNA, we then configured the system to detect lead and cadmium with the CadC aTF. Using this approach, we show the ability to detect lead and cadmium in water at concentrations own to 10 nM each (2 ppb for lead and 1 ppb for cadmium). Notably, these levels are below the EPA practical quantitation level of 5 ppb for lead

[0019] and EPA maximum contaminant level goal (MCLG) of 5 ppb for cadmium [2], and improve upon the LODs demonstrated by the ROSALIND cell-free biosensing platform by approximately two orders of magnitude [3], In addition, we show that these results can be obtained within 15 minutes (sample to answer < 15 min) with a digital readout. We anticipate these results will open the door to new approaches for rapid detection of w ater contaminants with platforms that can be embedded in portable digital devices.

[0092] Results

[0093] DNA-coated microcantilevers can detect transcription factor binding and unbinding in response to external ligands.

[0094] Our first goal was to test whether microcantilevers could detect transcription factor binding via coating their surface with DNA containing aTF operator sequences (FIG. 1A). To assemble this system, gold-coated microcantilevers were functionalized by immersing them in solution containing 5 pM thiolated double stranded DNA (dsDNA) that contained operator sequences corresponding to a specific aTF. As a proof-of-concept, we began with the well-characterized tetracycline-TetR repressor system

[0020] , We coated the microcantilevers using solution containing 5 pM thiolated dsDNA containing one tetO operator sequence within the 19 base pair dsDNA sequence (FIG. 1A).

[0095] DNA-coated microcantilevers were next placed inside a microfluidic chip (FIG. 2), and 10 pL of solution containing TetR at the specified concentration was added to the chip via pipette. Microcantilever bending was then detected optically using an AFM instrument as a proof-of- concept (see Methods). Exposing the microcantilever to a range of TetR concentrations ( 10 nM to 1 pM) increased bending magnitude over time, stabilizing within approximately 11 minutes of TetR addition for all TetR concentrations studied (FIG. IB). In addition, the stabilized bending magnitude increased with increasing TetR, up to a concentration of 1 pM, after which it saturated (FIG. IB). We therefore chose to proceed with the 1 pM TetR condition.

[0096] We next sought to investigate whether we could observe a change in microcantilever bending when the system is exposed to the TetR cognate ligand, anhydrotetracycline (aTc)

[0020] , To test this, DNA-coated microcantilevers were first exposed to 10 pL of 1 pM TetR for a period of approximately 15 min to allow complete bending, and then subjected to 10 pL of a solution containing either aTc or the control compound carbenicillin, which is known to not interact with TetR

[0021] , Notably, we did not observe a change in bending due to the addition of carbenicillin, indicating that TetR binding is not affected by the change in solution volume during this procedure. This allowed us to quantify the decrease in bending, denoted as ‘de-bending’, as the difference between the cantilever bending (deflection) in the presence of carbenicillin from that observed with a particular aTc condition at each point in time (see Methods) (FIG. 3). Using this approach, we observed an increase in de-bending over time, stabilizing within approximately 11 minutes of aTc addition (FIG. 1C). In addition, statistical analysis of de-bending trajectories showed that there are statistically significant differences in de-bending magnitude above baseline at 100 nM aTc under 5 min. Plotting the de-bending magnitude at 11 minutes versus aTc concentration showed an increase in de-bending over the aTc concentration range of 10 nM to 50 pM (FIG. ID).

[0097] Overall, this data demonstrated the ability of the DNA-coated microcantilever to detect the binding of aTFs, and their release upon exposure to cognate ligands within several minutes, demonstrating the speed of our system.

[0098] Varying DNA coating concentration influences microcantilever bending.

[0099] Next, we investigated how changing the concentration of DNA used to coat the microcantilevers influences microcantilever bending. For this study, we used solutions with varying concentrations of DNA containing one TetR operator sequence (IclO) within the 19 base pair DNA sequence to coat the microcantilevers (see Methods for full details on coating procedure). After coating the microcantilevers with DNA, 1 pM TetR protein solution was used to bend the microcantilevers as described in FIG. 1. The results show that using solution containing 5 pM DNA to coat the microcantilevers enables the maximum bending achieved (FIG. 13). This is the same DNA concentration that was used to coat the microcantilevers and obtain the results presented in FIG. 1. In addition, these results suggest that there is minimal variability in DNA attachment to the microcantilever surface because of the tight distribution we observe amongst replicates of microcantilevers coated using the same concentration of DNA, where the standard deviation in bending magnitude for each set of replicates is around 1 nm.

[0100] Varying DNA operator number and spacing enhances limit of detection.

[0101] Next, we sought to understand if modifying the DNA design by adding multiple operator sequences per dsDNA and altering the spacing between the operator sequences would influenceLOD. For the aTc-TetR system, we investigated one operator sequence per dsDNA (“IX”), two operator sequences per dsDNA with three base pair spacing between them (“2X3BP”). two operator sequences per dsDNA with six base pair spacing between them (“2X6BP”), and three operator sequences per dsDNA with six base pair spacing between them (“3X6BP”) (FIG. 5A).

[0102] Microcantilevers with different DNA designs were prepared and used for both bending and de-bending reactions as before. Increasing the number of operator sequences from one to two caused a greater magnitude of bending with a given TetR concentration, which was further increased by increasing the spacing of the two operator sequences from three to six bp (FIG. 5B). This translated into larger de-bending magnitude for aTc levels above 10 nM (FIG. 5C).

[0103] To compute the LOD for aTc, we chose the 2X6BP tetO DNA design with 1 pM TetR and performed a Student’s t-test to compare the de-bending magnitude at 11 minutes of three replicates of microcantilevers induced with aTc to three replicates of microcantilevers induced with the control compound (carbenicillin) (FIG. 5C). This t-test comparison of de-bending at 11 minutes, revealed that the 2X6BP design showed a statistically significant differences at 10 nM aTc compared to the control condition induced with carbenicillin. We were therefore able to define an LOD of 10 nM aTC using the 2X6BP tetO DNA design, which is notably lower than the LOD observed for the previously characterized ROSALIND transcription system of ~1 pM when using TetR to control the expression of a fluorescent RNA aptamer for detection, and -100 nM when using a negative feedback genetic circuit [3], Interestingly, further improvements in bending and de-bending magnitudes were not observed for the 3X6BP DBA design, suggesting that there is a limit to this approach of tuning.

[0104] Next, we investigated the shelf-stability of the DNA coated microcantilevers using the 2X6BP tetO DNA design with 1 pM TetR. After coating the microcantilevers with the 2X6BP tetO DNA, the microcantilevers were left at room temperature for 0. 15. or 30 days before performing de-bending studies using 1 pM TetR and inducing with 0.01. 1. or 50 pM aTc (FIG. 5D). Results show consistency in de-bending magnitude at 11 minutes over the thirty-day study period. The only statistically significant difference in de-bending magnitude at 11 minutes occurred between Day 0 and Day 30 at 1 pM aTc induction, wherein a decrease in average de-bending of 16% occurred (P = 0.02).

[0105] Finally, we evaluated the selectivity of the system using the 2X6BP tetO design with 1 pM TetR. Microcantilevers were induced with chloramphenicol or kanamycin at either 1 pM or 50 pM. Results show that induction with these other antibiotics does not lead to appreciable de- bending at 11 minutes, demonstrating the selectivity of the TetR system tetracycline-like antibiotics (FIG. 5E).

[0106] These results demonstrate that DNA design is an additional handle with which the aTF- microcantilever system can be tuned, and that the system has improved LODs over cell-free gene expression systems that utilize the same aTFs.

[0107] Single digit parts per billion detection of Pb2+and CD2+can be achieved with microcantilevers interfaced with the CadC transcription factor.

[0108] We next sought to extend our sensor platform to the detection of heavy metals that can lead to serious health consequences if present in drinking water [22, 23], Lead is of particular concern, and lead concentration is tightly regulated by the US EPAs Lead and Copper Rule (LCR)

[0019] , The LCR sets an action level of 10 parts per billion (ppb, 48 nM) for lead and stipulates that water systems must take action via public education or corrosion treatment if the water system’s 90thpercentile lead sampling result exceeds this level

[0024] ,

[0109] To configure the microcantilever system to detect lead Pb2+, we utilized the well- characterized transcription factor CadC from Staphylococcus aureus [25, 26], which has been previously used in the ROSALIND system [3], CadC also has the advantage of being able to detect Cd2+[26, 27] and does so through a similar mechanism as TetR, except it recognizes a different 22 bp DNA operator sequence CadO

[0028] , We replaced tetO with the cadO operator sequence in the 2X6BP DNA template design and characterized bending with respect to the addition of CadC and either lead or cadmium (FIGS. 6 and 7). Similar to microcantilever bending with TetR (FIG. 1 A), our results show that microcantilever bending magnitude increases with increasing CadC concentration from 10 nM to 5 pM after which it plateaus at higher concentrations (FIGS. 6 and 7). In addition, the bending magnitude stabilizes within approximately 11 minutes of CadC addition for all CadC concentrations tested (FIGS. 6 and 7). The optimized concentration of 5 pM CadC was therefore chosen to obtain maximum microcantilever bending prior to ligand (lead or cadmium) addition.

[0110] We next studied de-bending when subjecting CadC-bound microcantilevers to either Pb2+or Cd2+(FIGS. 8B-8C). In both cases, we observed an increase in de-bending response at 11 minutes when subjected to increasing concentrations of Pb2+and Cd2+. To compute the LOD for Pb2+and Cd2+, we used the 2X6BP cadO system with 5 pM CadC and performed a Student’s t-test to compare de-bending magnitude at 11 minutes for three replicates of microcantilevers induced with Pb2+or Cd2+to three replicates of microcantilevers induced with the control compound (carbenicillin) (FIGS. 8B, C). This t-test comparison of de-bending at 11 minutes revealed that the 2X6BP cadO system showed a statistically significant difference at 10 nM Pb2+and 10 nM Cd2+compared to the control condition induced with carbenicillin. We were therefore able to define an LOD of 10 nM (2 ppb) for Pb2+and 10 nM (1 ppb) for Cd2+, both lower than the EP A action limitsof 10 ppb and 5 ppb for lead and cadmium [2, 19], respectively. Notably these values were two orders of magnitude lower than those demonstrated previously with ROSALIND, which were 1.2 pM (250 ppb) for Pb2+and 1.25 pM (140 ppb) for Cd2+[3], These results demonstrated that the aTF -microcantilever platform provides a much lower LOD than the current versions of solutionbased aTF detection.

[0111] Next, we investigated the shelf-stability of the DNA coated microcantilevers using the 2X6BP cadO DNA design. After coating the microcantilevers with the 2X6BP cadO DNA, the microcantilevers were left at room temperature for 0, 15, or 30 days before performing de-bending studies using 5 pM CadC and inducing with 0.01, 1, or 50 pM Pb2+(FIG. 8D). Results show that de-bending magnitude decreased by an average of 3.54 nm over the course of the 30-day study.

[0112] Finally, we evaluated the selectivity of the system using the 2X6BP cadO DNA design with 5 pM CadC. Microcantilevers were induced with Zn2+(ZnSCL) or Cu2+(CuCb) at either 1 pM or 50 pM. Results show that induction with these other metals does not lead to appreciable de- bending, demonstrating the selectivity of the system (FIG. 8E).

[0113] CadC-interfaced microcantilevers can be used to detect lead in drinking water.

[0114] To understand the ability of aTF-interfaced microcantilevers to detect contaminants in drinking water, we performed a study using the CadC system with 5 pM CadC and the 2X6BP cadO DNA design. In this study, unfiltered, non-pretreated drinking water was spiked with vary ing amounts of PbCh. After bending the microcantilevers with 10 pL of 5 pM CadC solution. 10 pL of spiked drinking water samples were added, and de-bending was measured as described in FIG. 8B. The spiked drinking water samples were also analyzed via graphite furnace atomic absorption spectrometry (GFAAS) as a gold standard method to determine the concentration of Pb in the samples. Importantly, the unspiked drinking water contained 0.25 ppb Pb (1.2 nM) per GFAAS measurement. The results demonstrate that a similar de-bending response can be obtained in drinking water to that which was obtained in 18 MOhm water (Figure 14A). We also computed a line of best fit to correlate logl0([Pb] / pM) and de-bending magnitude on the range 0.001 - 0. 1 pM Pb for the studies performed in 18 MOhm water (FIG. 14B). This line of best fit correlated w ell with the de-bending studies performed on the drinking water samples (FIG. 14C). We then used this line of best fit to predict the concentrations of Pb2* in the spiked drinking water samples based on the de-bending magnitudes we measured (FIG. 14D). These predictions correlate well with the results obtained via GFAAS.

[0115] Discussion

[0116] In this work, we successfully merged cell-free biosensing systems with nanomechanical detection systems to create a new platform for the rapid and sensitive detection of chemicalcompounds. By coating microcantilevers with DNA containing the operator sequences of aTFs, we were able to achieve 10 nM LOD for anhydrotetracycline, lead, and cadmium within ~11 minutes (FIGS 5, 8). This represents concentrations of 4 ppb for anhydrotetracycline, 2 ppb for lead, and 1 ppb for cadmium, which are approximately two orders of magnitude lower than what has been achievable with cell-free biosensing systems that use these aTFs to control reporter gene expression as an output [3], In addition, the nanomechanical system is as fast as cell-free transcriptional biosensors that use DNA strand displacement read outs [4],

[0117] Importantly, our LOD of 2 ppb for lead aligns with the Illinois Department of Public Health’s lead testing requirement for standard analytical chemistry' techniques, which states that the minimum detection limit of a chosen analytical testing method should not exceed 2.0 ppb

[0029] , Our LOD for lead is also below the EPA action level of 10 ppb, which stipulates that water systems must take action via public education or corrosion treatment if the water system’s 90thpercentile lead sampling result exceeds this level

[0024] , Our limit of detection for cadmium is below the EPA maximum contaminant level goal (MCLG) of 5 ppb for cadmium [2], which describes the level of a contaminant in drinking water below which there is no known or expected risk to health.

[0118] While the microcantilever system has superior sensing abilities over previously reported cell-free biosensing systems, there are several additional advantages and some potential disadvantages of the system. In terms of advantages, microcantilevers address another limitation of cell-free biosensing systems which is the lack of quantification. While genetic circuits can be added to cell-free biosensing systems to produce devices that yield semi-quantitative read outs [4], they do not produce digital read outs and thus cannot offer the same level of quantification that a microcantilever system coupled with on-board processing can. In fact, if on-board data processing is used, microcantilever systems could be used to deliver results even faster by using short time signal transients to predict outputs signals. In addition, microcantilever devices can be easily multiplexed

[0018] , potentially enabling several chemicals to be detected simultaneously. However, these advantages may come at the additional cost of manufacturing and deploying microcantilever systems, compared to the relative ease of manufacturing and distributing single-use cell-free biosensing systems

[0010] ,

[0119] While the microcantilever and cell-free biosensing systems utilize the same transcription factor-DNA operator pairs to detect chemicals, we observed that they appear to have different sensing properties. Specifically, the dose-response curves obtained using the microcantilever platform did not contain a characteristic sharp inflection point that is typical of the dose-response curves obtained using the cell-free gene expression systems [3], To investigate this observation, we constructed a simple double-equilibrium model of the anhydrotetracyclme-TetR system withthe IX tetO DNA design (see Example 2). We found that the model can reproduce results obtained via the ROSALIND cell-free setup [3] when the association constants available in the literature for the anhydrotetracycline-TetR system were used. However, reasonable agreement between the model predictions and the microcantilever dose-response curves required that the literature association constants between aTF and DNA be modified by several orders of magnitude and the initial TetR concentration be lowered by two orders of magnitude. This seems to indicate that the physics of aTF-DNA binding / un-binding on a two-dimensional coated surface is causing a significant perturbation to the biochemistry of aTF-DNA binding, though more work is needed to understand the mechanistic details of these differences.

[0120] We also note that this work is similar in spirit to recent work that couples cell-free biosensors with portable glucose meters for multiplexed, digital pathogen detection

[0012] , To achieve digital readout from single-use cell-free biosensors, Amalfitano et al. utilized genetic circuits to transform the outputs of transcriptional biosensors into a glucose signal via glucogenic enzy mes. The enzymatically generated glucose can then be read by low-cost glucose meters for digital sensing. This strategy’ would be broadly applicable to many transcription-based cell-free biosensing platforms, including those that leverage aTFs for analyte detection. In contrast to transcription-based cell-free biosensing platforms, the microcantilever-based sensing strategy outlined here directly converts aTF binding events into digital output, with the potential for continuous readout when the microcantilevers are coupled with MOSFET technology. The work outlined here thus employs a fundamentally different approach to signal transduction and reporting, providing a complementary solution to single-use biosensors that involve cell-free gene expression.

[0121] This work is similar in spirit to previous work by HF Ji et al, who describe microcantileverbased methodologies for detecting Pb2+and Cd2+[36-38] including DNA-coated microcantilevers

[0037] and antibody-modified microcantilevers

[0038] , Such approaches have been able to demonstrate impressive LODs including 10 nM for Pb2

[0037] and 1 nM for Cd2+

[0038] , The feature that distinguishes this work from previous studies is the coupling of aTF proteins with microcantilevers, which we believe builds upon the successes of previous work and can expand the types of chemicals that can be detected via microcantilevers as aTF proteins have been documented to detect a wide range of chemical species including both metals and also many small organic molecules including antibiotics among other biologically important chemicals [3],

[0122] In addition, the aTF-interfaced microcantilever sensing strategy outlined here may provide advantages over other sensing strategies by enabling plug and play sensing, where DNA sequences and aTFs are simply interchanged to detect different analytes. Fortunately, numerous aTFs havebeen characterized to date including those that sense metals as well as uncharged small molecules [3]. enabling the detection of a wide range of compounds. In fact, over 200.000 aTFs have been identified in the TetR family alone

[0021] , In contrast, many electrochemical methods such as anodic stripping are limited to the detection of metals, although such methods can achieve LODs on the order of 0. 1 ppb

[0039] , which are lower than the single digit ppb LODs demonstrated in this work. Other electrochemical methods have been developed to detect organic and biological molecules; however, these methods typically require electrodes, pretreatments, and reaction conditions that are specifically tailored to analyte of interest [40, 41], while the aTF interfaced microcantilevers described here demonstrate the ability to detect markedly different chemical species including antibiotics (anhydrotetracycline) and metal cations (Pb2+Cd2+) simply by interchanging the aTF and DNA sequences used.

[0123] Overall, the merging of cell-free biosensors and microcantilever sensing represents a promising new platform that provides substantial value by enabling diagnostic systems that can address important public health challenges such as measuring water quality [1]. The ability7for MOSFET devices to implement rapid and sensitive detection of water contaminants is particularly promising, as these systems can be embedded in integrated circuits

[0015] , potentially enabling their deployment in portable devices, and can be multiplexed [17, 18] potentially allowing the detection of multiple contaminants simultaneously. Furthermore, the microcantilever sensing framework should be readily translatable to sensing other chemicals for which aTFs are available

[0030] , Finally, incorporation of on-board data processing can further be used to process and communicate data on-the-fly, helping to make the system more robust

[0031] and enabling large-scale data-driven approaches to addressing public health challenges [1], With further development, we envision that this system can be combined with MOSFET technology

[0015] to pave the way for widespread incorporation of this platform within portable digital devices.

[0124] Materials and Methods

[0125] Synthesis of allosteric transcription factors (aTFs)

[0126] Expression and purification of the TetR and CadC aTFs was performed as described in [3], Briefly, for TetR expression and purification, sequence-verified pET-28c plasmids were transformed into the Rosetta 2(DE3) pLysS E. coli strain for protein expression. Cultures were grown up and then induced for overexpression with isopropyl [3-d- 1 -thiogalactopyranoside. The cultures were then pelleted via centrifugation, resuspended in lysis buffer (10 mM Tris-HCl, pH 7.5-8.5, 500 mM NaCl, 1 mM tris(2-carboxyethyl)phosphine (TCEP) and protease inhibitor (complete EDTA-free Protease Inhibitor Cocktail, Roche)), and then lysed via ultrasonication. Lysates were then centrifuged, and the supernatant purified using His-tag affinity chromatographyfollowed by size-exclusion chromatography. Collected proteins were concentrated and buffer exchanged (25 mM Tris-HCl. 100 mM NaCl, 1 mM TCEP, 50% glycerol, v / v) via centrifugal filtration. Protein concentrations were measured via the Qubit Protein Assay Kit, and protein purity and size were verified via SDS-PAGE gel. Purified proteins were stored at -20 °C. For CadC expression and purification, the exact same steps were followed as for TetR with the exception that the pET-3d plasmid backbone was used, and a higher concentration of TCEP (5 mM) was used in the buffers. ATFs were diluted from stock to their indicated working concentrations with 18 MOhm w ater.

[0127] DNA coating of microcantilevers

[0128] All DNA oligos (Table 1) w ere purchased from Integrated DNA Technologies (IDT), using either standard desalting or HPLC purification as indicated. DNA oligo stock solutions were made by dissolving DNA oligos from IDT in MilliQ water to yield a concentration of 500 pM. To generate w orking dsDNA solutions for microcantilever coating, equimolar ratios of 5’ C6 thiol modified DNA oligos (500 pM stock) and their complements (500 pM stock) were combined along with 5X annealing buffer (500 mM potassium acetate, 150 mM HEPES, pH 8.0) and diluted to the proper final volume using MilliQ water. The solutions were then annealed by heating to 95 °C for 3 minutes inside a heat block and then allowing them to cool at room temperature for 1 hour. 5 pM dsDNA solution was chosen for microcantilever coating based on experimentation with variable concentrations of dsDNA (FIG. 13). Gold-coated microcantilevers purchased from Nanoworld Innovative Technologies (Product # ARROW-TL1 Au-50), were then placed inside the wells of a 96 well plate containing the 5 pM dsDNA oligo solution along with 500 pM TCEP (one microcantilever per well). TCEP was added at a ratio of 100: 1 TCEP:DNA in accordance with IDT’s protocol for reduction for oligonucleotides with thiol modifications. The microcantilevers were then left to stand for approximately two hours at room temperature. The microcantilevers were then retneved from the wells, air dried and used for analyte detection.

[0129] Shelf Stability Study

[0130] Microcantilevers were coated with double-stranded DNA as described in the “DNA coating of microcantilevers’' section. After coating with double-stranded DNA, microcantilevers were stored in a plastic case inside a cabinet at room temperature for either 0, 15, or 30 days before performing de-bending reactions as described in the “AFM deflection measurement for bending / de-bending reactions” section.

[0131] With the dimensions of the microcantilevers used in these Examples, the estimated packing density is a range of about 5.2 x 1012 / cm2to about 9.8 x 1013 / cm2. The surface stress values that we acquired also validate the packing density within this range, ft should be noted that thequantitative measurement of DNA packing density on a microcantilever is influenced by various factors, and specific numerical values can vary based on experimental conditions and methodologies. Typically, DNA packing density is expressed in terms of the number of DNA molecules per unit area. The length of the DNA which we have used in these Examples is around 0.34 nm per base, while the diameter is around 2 nm. The actual values can range widely depending on the length of the DNA strands, their conformation, and the surface functionalization techniques used.

[0132] Table 1 : DNA Oligos used in microcantilever experiments. aTF operator sites are underlined, with base pair spacers italicized.

[0133] Microfluidic chip design and manufacture

[0134] Microfluidic chip design and manufacture followed previously published protocols [16, 32], Microfluidic chips were designed and manufactured by erstwhile Nanoink Inc in 2006. These microfluidic chips were employed for detection purposes in our earlier published work [16, 32], Briefly, the microfluidic chips, essential components in microscale experiments, were intricately fabricated through photolithography and etching by Nanoink Inc in 2006.

[0135] AFM deflection measurement for bending / de-bending reactions

[0136] For each bending reaction, a DNA-coated microcantilever was placed inside a fabricated microfluidic chip, which was then placed inside an atomic force microscope system (Bruker High Performance Bioscope Resolve Life Science Imaging System). The laser was aligned manually, and measurement initiated. 10 pL of allosteric transcription factor solution was then added to the to the microfluidic chip via pipette (see synthesis of allosteric transcription factors for details on aTF working solution preparation). Microcantilever deflection was then recorded at 1 -minute intervals for a 15-minute period. For each de-bending reaction, a microcantilever was first bent using 10 pL of aTF solution (1 pM TetR or 5 pM CadC) as described in the procedure above. The 1 pM TetR and 5 pM CadC solutions used for de-bending studies were the same solutions used in the bending studies. Next, 10 pL of solution containing a specified ligand or control compound at the indicated concentration was added to the microfluidic chip. To control for the effect of the added ligand solution diluting the aTF concentration and potentially causing de-bending, we performed experiments with the non-cognate ligand carbenicillin which is not recognized by the aTFs used in this study. Microcantilever deflection was then recorded at 1 -minute intervals for a 15-minute period.

[0137] Calculation of microcantilever bending and de-bending

[0138] The measurement of microcantilever deflection was used to calculate bending and de- bending in different experimental conditions (FIGS. 1 and 3). In the presence of aTF alone, bending was calculated as being equal to microcantilever deflection (FIGS. 1 and 3).

[0139] The calculation of de-bending combined deflection measurements in the presence of cognate aTF ligands and control compounds and was calculated as the difference in bending (deflection) between the sensing microcantilever, induced with cognate aTF ligand, and the average bending of the control microcantilevers (FIGS. 1 and 3).

[0140] The calculation of standard deviation (o) for use in error shading for bending experiments (ex: FIG. IB) was computed using the definition of standard deviation for n measurements (Scheme 1), while the calculation of standard deviation (OA-B) for use in error shading for de- bending experiments (ex: FIG. 1C) was computed using error propagation for de-bendingexperiments (Scheme 1).

[0141] Scheme 1. Calculating Microcantilever Bending, De-Bending, and Standard Deviation. Cantilever deflection is the measurement of microcantilever deflection in the presence of cognate ligand, control cantilever deflection is the measurement of microcantilever deflection in the presence of a control compound taken at the same time point as the microcantilever deflection, and n is equal to the number of control microcantilevers used. Where crAis variance of deflection measurements at a given time in the presence of cognate ligand,is variance of deflection measurements in the presence of the control compound, andis their covariance.

[0142] Bending = cantilever deflection r / A , / -. -I r-x i i . z .i i n - z x X n control cantilever deflection(t)

[0143] Cantilever De — bending(t) = cantilever deflection(t) -

[0144] The calculation of standard deviation (s) for use in error shading as evident in FIG. IB was computed as follows for bending experiments: n cantilever deflection(t) — mean cantilever deflection(t)

[0145] The calculation of standard deviation (CTA-B) for use in error shading as evident in Fig. 1C was computed as follows for de-bending expenments:

[0146] Drinking Water Studies

[0147] A municipal drinking water sample was harvested from a home in Evanston, IL and stored at room temperature inside a sealed 1000 mL high-density polyethylene screw-top container. To prepare spiked drinking water samples, 100X stock solutions of PbCL were prepared in 18 MOhm water and then added at 1:99 volume: volume to the drinking water to yield IX final concentration. For example, 100 pL of 10 pM PbCh solution was added to 9.9 mL drinking water to yield 10 mL of 100 nM spiked drinking water. A portion of each spiked drinking water sample was set aside for GFAAS measurement, while the balance was used to perform de-bending studies. For de- bending studies, microcantilevers were first bent with 10 pL of 5 pM CadC in 18 MOhm water as described previously. Then, as described previously. 10 pL of drinking water sample was added and microcantilever bending (deflection) was recorded.

[0148] Dissolved Pb analysis by GFAAS

[0149] Spiked samples were acidified to pH ~1 with ultrapure HNO3 (Fisher Chemical, catalog no. CAS7697-37-2). Concentrations were determined using a ContrAA 800G high-resolution continuous source graphite furnace atomic absorption spectrometer (HR-CS-GFAAS) instrument(Analytik Jena) equipped with a transversely heated graphite tube atomizer. The analyses were performed using the 283.306 nm wavelength for lead and NH4H2PO4 (High Purity Standard, catalog SKU: MM-9002-1) as a matrix modifier. Calibration solutions were prepared from a Spex Certi-Prep Pb standard - 1000 pg / mL - and ranged from 2 pg / mL to 10 pg / mL. Quality control was assessed using the AQUA-1 drinking water certified reference material from the national research center Canada. Based on the calibration curve presented in FIG. 15, the reported value of the AQUA-1 Pb concentration was 1.361 + / - 0.054 pg / mL for a certified value of 1.364 + / - 0.034 pg / mL. Three replicate analyses were performed for standards and samples that were averaged, and uncertainties were determined from the calibration curve (FIG. 15, Table 2).

[0150] Table 2. GFAAS measurements for drinking water spiked with varying amounts of Pb2+, the results of which are presented in FIG. 14.

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[0152] Example 2 - Mechanistic Model of Cantilever-based Detection Sensitivity Improvement

[0153] Model Setup

[0154] The items below7describe the double-equilibrium model developed for the anhydrotetracycline-TetR system with the IX DNA design (FIG. 9) to investigate the differences between the dose-response curves obtained in this work and those obtained via the ROSALIND setup [3], See FIG. 9 for an overview of the double equilibrium model of transcription factor binding to DNA and target compound. The variables are summarized in Table 3. The variables are assigned values according to literature values, experimental conditions, or best estimates as indicated.

[0155] Table 3. Description of elements of the tetracycline-TetR model system.

[0156] In this model we assume that TetR exists only as a dimen two anhydrotetracycline molecules bind to one TetR dimer, a TetR dimer bound to anhydrotetracycline cannot bind DNA, and there is only one tetO site per DNA.

[0157] Table 4. Parameters used in the double-equilibrium model.

[0158] Equations:

[0159] The definitions of the equilibrium dissociation constants are as follows:

[0160] The equilibrium dissociation constant equations can then be incorporated into the species conservation equations for total DNA. aTC and TetR. Note, because the DNA is deposited on the cantilever surface, DNA concentrations are in moles per unit area, while moles per unit volume are used for TetR and aTC because those species can be released in the solution surrounding the cantilever. Also note that because one TetR dimer binds two anhydrotetracycline molecules, the notation (aTC)2 is used. Total (aTC)2 in the system is simply the total aTC concentration in the system divided by two.Eq. 5) [TetR]0V = [TetR]V + [(aTC)2■ TetR]V + [DNA ■ TetR]A = [TetR]V + [(aTC)2][TetR]v[DNA][TetR]AKD,L KD 1

[0161] Solving the double equilibrium

[0162] For a given experimental condition (specified by the input concentration of each molecular species), the fraction of DNA unbound by TetR is proportional to the output signal generated. Therefore, the goal of the double equilibrium analysis is to solve for [DNA] / [DNA]o as a function of [TetR]o and [(aTC)2]o. Rearranging Eq. (5), and writing [(aTC)2] and [DNA] in terms of [TetR] using Eq. (4) and Eq. (3), respectively, yields:

[0163] Eq. (6) can be solved numerically for [TetR], which can then be plugged into a rearranged version of Eq. (3) to find:>>

[0164] For the ROSLAND system, all concentrations are on a volume basis, so Eq. (6) is re-written as:

[0165] Making use of the fact that the volume term cancels. Solving Eq. (8) numerically can then be used in Eq. (7) in the same way.

[0166] Analysis of ROSALIND sensitivity

[0167] We first explored the ability of the model to reproduce the results obtained using the ROSALIND setup [3], Specifically, we numerically solved equation (8) for the case of 25 nM DNA template and 1250 nM TetR, with other parameters specified as indicated in Table 4. Resultsfrom this model are in good agreement with those from experiment, with the model accurately predicting the aTC concentration at which the system activates (FIG. 10).

[0168] Analysis of microcantilever sensitivity

[0169] We next used the double-equilibrium model to investigate the predicted dose-response curve for the microcantilever setup using parameter values from Table 4. Because the concentration of DNA immobilized on the microcantilever surface is unknown, we chose to evaluate two different DNA concentrations corresponding to ten times the maximum feasible concentration (F = 10) and one one-hundredth the maximum feasible concentration (F = 0.01). For the two DNA concentrations studied, the dose response curves predicted by the model are nearly identical. This is because in both cases, TetR is present in great excess compared to DNA, or [TetR]0» ibecause » p The model’s predictions are also of much different shape[DNA]0KD LH' compared to the dose-response curves obtained via the microcantilever setup, although the EC50 values for the model and the data are generally within an order of magnitude of each other (FIG. 11).

[0170] We next explored if closer agreement between the model’s dose-response output and debending data could be achieved by modifying the values of the variables in the model. Using the maximum feasible surface DNA concentration (F = 1), closer agreement between the model’s dose-response output and de-bending data required lowering the initial concentration of TetR ([TetR]0) by approximately two orders of magnitude (from 500 nM to 5 nM) and lowering the TetR- / e / (9 dissociation constant (KD 1) by approximately four orders of magnitude (from 1.79e-10 to 2.27e-14 M) (FIG. 12). Lowering KDwas required to achieve a shallower dose-response slope compared to the step-like response predicted using KD ;1from literature, while lowering [TetR]0was required to shift the EC50 value to better align with those of the de-bending data. While the concentration of aTF is indeed lowered during de-bending (10 pL of ligand solution is added to the existing 10 pL of aTF solution used for bending), this twofold decrease in aTF concentration is not nearly sufficient to account for the hundred-fold decrease in aTF concentration required for the model to better agree with the data.

[0171] The results suggest that the microcantilever sensing system behaves differently from the ROSALIND sensing system [3], even though both systems employ the same aTF-based sensing strategy. One possible source of difference is signal propagation; in the case of the microcantilever, aTF binding and unbinding is transduced into surface stress and thus cantilever deflection, while for ROSALIND, aTF binding and unbinding is transduced into transcription and thus fluorescent aptamer formation.

[0172] It is also possible that aTF-DNA interactions are altered in the microcantilever setup, where DNA is immobilized on the microcantilever surface as opposed to diffusing freely in solution. Immobilizing DNA on the microcantilever surface could influence aTF-DNA interactions if the immobilized DNA forms a dense forest on the microcantilever surface that affects the abil i ty of aTF to bind / unbind from the DNA, or if DNA structural flexibility' is altered by its immobilization. The environment around the cantilever may also differ substantially from that of the bulk as it would be expected that the immobilized DNA would result in a highly localized negative charge that would need to be countered by the presence of other cations in the area, possibly resulting in a localized area of high ionic strength.

[0173] References1. Agarwal, D.K., A.C. Hunt, G.S. Shekhawat, L. Carter, S. Chan, K. Wu, L. Cao, D. Baker, R. Lorenzo-Redondo, E.A. Ozer, L.M. Simons, J.F. Hultquist, M.C. Jewett, and V.P. Dravid, Rapid and Sensitive Detection of Antigen from SARS-CoV-2 Variants of Concern by a Multivalent Minibinder-Functionalized Nanomechanical Sensor. Anal Chem, 2022. 94(23): p. 8105-8109. Available from: https: / / www.ncbi.nlm.nih.gov / pubmed / 35652578.2. Agarwal, D.K., V. Nandwana. S.E. Henrich. V. Josyula, C.S. Thaxton, C. Qi, L.M.Simons, J.F. Hultquist, E.A. Ozer, G.S. Shekhawat, and V.P. Dravid, Highly sensitive and ultrarapid antigen-based detection of SARS-CoV-2 using nanomechanical sensor platform. Biosens Bioelectron, 2022. 195: p. 113647. Available from: https : / / www. ncbi . nlm. nih. gov / pubmed / 34583103.3. Jung, J.K., K.K. Alam, M.S. Verosloff, D.A. Capdevila, M. Desmau, P.R. Clauer, J.W. Lee, P.Q. Nguyen, P.A. Pasten, S.J. Matiasek, J.F. Gaillard, D.P. Giedroc, J.J. Collins, and J.B. Lucks, Cell-free biosensors for rapid detection of water contaminants. Nat Biotechnol, 2020. 38(12): p. 1451-1459. Available from: https: / / www.ncbi.nlm.nih.gov / pubmed / 32632301.4. Kamionka, A., J. Bogdanska-Urbaniak. O. Scholz, and W. Hillen. Two mutations in the tetracycline repressor change the inducer anhydrotetracycline to a corepressor . Nucleic Acids Res, 2004. 32(2): p. 842-7. Available from: https: / / www.ncbi.nlm.nih.gov / pubmed / 14764926.5. Kedracka-Krok, S. and Wasylewski, Z., Kinetics and equilibrium studies of Tet repressoroperator interaction. J Protein Chem, 1999. 18(1): p. 117-25. Available from: https: / / www.ncbi.nlm.nih.gOv / pubmed / l 0071936.6. Lederer, H., R.P. May, J.K. Kjems, G. Baer, and H. Heumann, Solution structure of a short DNA fragment studied by neutron scattering. Eur J Biochem, 1986. 161(1): p. 191-6. Available from:

[0174] Additional Sequences

[0175] TetR (SEQ ID NO: 13):MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSKLAAALE

[0176] CadC (SEQ ID NO: 14):MKKKDTCEIFCYDEEKVNRIQGDLQTVDISGVSQILKAIADENRAKITYALCQDEELCVCDIANILGVTIANASHHLRTLYKQGVVNFRKEGKLALYSLGDEHIRQIMMIALAHKKEVKVNV

Claims

CLAIMSWhat is claimed is:

1. A system for detecting one or more analytes in a liquid sample, the system comprising: a microcantilever; a plurality of polynucleotides coupled to at least a portion of the microcantilever, wherein each of the polynucleotides comprises one or more operator sequences; and one or more allosteric transcription factors, wherein each of the one or more allosteric transcription factors binds to at least one of the one or more operator sequences and at least one of the one or more analytes; wherein, when the one or more allosteric transcription factors bind to the one or more operator sequences, the microcantilever bends; and wherein, when the one or more analytes bind to the one or more allosteric transcription factors, the one or more allosteric transcription factors cannot bind to the one or more operator sequences.

2. The system of claim 1, wherein each of the polynucleotides are double stranded DNA (dsDNA).

3. The system of claim 2, wherein the dsDNA is thiolated dsDNA.

4. The system of any one of claims 1-3, wherein the microcantilever is at least partly coated in gold.

5. The system of any one of claims 1-4, wherein each of the polynucleotides comprises at least two operator sequences.

6. The system of claim 5, wherein the two operator sequences are separated by a spacer sequence.

7. The system of claim 6, wherein the spacer sequence is between about 2 nucleotides and about 30 nucleotides.

8. The system of claim 6, wherein the spacer sequence is about 6 nucleotides.

9. The system of any one of claims 1-7, wherein each polynucleotide comprises a sequence having at least 90% identity to one of SEQ ID NOs: 1-12.

10. The system of any one of claims 1-9, wherein the microcantilever has a maximum dimension of between about 100 pm and about 1.5 mm.

11. The system of any one of claims 1-10, wherein the microcantilever has a length of between about 450 pm to about 520 pm, a width of between about 80 pm to about 100 pm, and a thickness of between about 1 pm to about 1.5 pm.

12. The system of any one of claims 1-11, wherein the microcantilever comprises a silicon- based material.

13. The system of any one of claims 1-12, wherein the one or more allosteric transcription factors compnse at least one of TetR and CadC.

14. The system of claim 12, wherein the one or more allosteric transcription factors comprise CadC, and wherein the one or more analytes comprise at least one of Pb2+and Cd2+.

15. The system of claim 13, wherein the system can detect Pb2+in the liquid sample at a concentration of less than 10 parts per billion (ppb); and wherein the system can detect Cd2+in the liquid sample at a concentration of less than 10 ppb.

16. The system of claim 12, wherein the one or more allosteric transcription factors comprise TetR, and wherein the one or more analytes comprise anhydrotetracycline (aTc).

17. The system of claim 13, wherein the system can detect aTc at a concentration of less than 1 pM.

18. The system of any one of claims 1-17, wherein the microcantilever is in a microfluidic chip.

19. The system of any one of claims 1-18, further comprising: at least one additional microcantilever:a plurality of additional polynucleotides coupled to at least a portion of the at least one additional microcantilever, wherein each of the additional polynucleotides comprises one or more additional operator sequences; and one or more additional allosteric transcription factors, wherein each of the one or more additional allosteric transcription factors binds to at least one of the one or more additional operator sequences and at least one additional analyte; wherein, when the one or more additional allosteric transcription factors bind to the one or more additional operator sequences, the at least one additional microcantilever bends; wherein, when the additional analytes bind to the one or more additional allosteric transcription factors, the one or more additional allosteric transcription factors cannot bind to the one or more additional operator sequences; and wherein the additional operator sequences of the polynucleotides coupled to each additional microcantilever are different.

20. A method for detecting one or more analytes in a liquid sample using the system of any one of claims 1-19.

21. A method for detecting one or more analytes in a liquid sample, the method comprising: contacting an allosteric transcription factor interfaced microcantilever to the sample; and detecting a de-bending of the microcantilever; wherein the allosteric transcription factor interfaced microcantilever comprises: a plurality' of polynucleotides coupled to at least a portion of the microcantilever, wherein each of the polynucleotides comprises one or more operator sequences; and one or more allosteric transcription factors bound to the one or more operator sequences; wherein, when the one or more analytes bind to the one or more allosteric transcription factors, the one or more allosteric transcription factors are released from the one or more operator sequences; and wherein detection of de-bending indicates the presence of the one or more analytes.

22. The method of claim 21 , wherein detecting the de-bending comprises utilizing atomic force microscopy or an electrical detection method.

23. The method of claim 21 or 22, wherein the contacting is done for between about 15 minutes and about 30 minutes.

24. The method of any one of claims 21-23, wherein each of the polynucleotides are double stranded DNA (dsDNA).

25. The method of claim 24, wherein the dsDNA is thiolated dsDNA.

26. The method of any one of claims 21-25, wherein the microcantilever is at least partly coated in gold.

27. The method of any one of claims 21-26, wherein each of the polynucleotides comprises at least two operator sequences.

28. The method of claim 27, wherein the two operator sequences are separated by a spacer sequence.

29. The method of claim 28, wherein the spacer sequence comprises between about 2 nucleotides to about 30 nucleotides.

30. The method of any one of claims 21-29, wherein each polynucleotide comprises a sequence having at least 90% identity to one of SEQ ID NOs: 1-12.

31. The method of any one of claims 21-30, wherein the one or more allosteric transcription factors compnse at least one of TetR and CadC.

32. The method of claim 31, wherein the one or more allosteric transcription factors comprise CadC, and wherein the one or more analytes comprise at least one of Pb2+and Cd2+.

33. The method of claim 32, wherein Pb2+in the sample can be detected at a concentration of less than 10 parts per billion (ppb); and wherein Cd2+in the liquid sample can be detected at a concentration of less than 10 ppb.

34. The method of claim 31, wherein the one or more allosteric transcription factors comprise TetR, and wherein the one or more analytes comprise anhydrotetracycline (aTc).

35. The method of claim 34, wherein aTc in the sample can be detected at a concentration of less than 1 pM.

36. The method of any one of claims 21-35. wherein the microcantilever has a maximum dimension of between about 100 pm and about 1.5 mm.

37. The method of any one of claims 21-36, wherein the microcantilever has a length of between about 450 pm to about 520 pm, a width of between about 80 pm to about 100 pm, and a thickness of between about 1 pm to about 1.5 pm.

38. The method of any one of claims 21-37, wherein the microcantilever comprises a silicon- based material.

39. The method of any one of claims 21-38, wherein the allosteric transcription factor interfaced microcantilever is in a microfluidic chip.

40. The method of any one of claims 21-39, wherein the liquid sample comprises water or a beverage.

41. The method of any one of claims 21-40, further comprising: contacting at least one additional allosteric transcription factor interfaced microcantilever to the sample; and detecting a de-bending of the additional microcantilever; wherein each of the additional allosteric transcription factor interfaced microcantilevers comprises: a plurality of additional polynucleotides coupled to at least a portion of the additional microcantilever, wherein each of the additional polynucleotides comprises one or more additional operator sequences; and one or more additional allosteric transcription factors bound to the one or more additional operator sequences; wherein, when one or more additional analytes bind to the one or more additional allosteric transcription factors, the one or more allosteric transcription factors are released from the one or more additional operator sequences;wherein detection of de-bending indicates the presence of the one or more additional analytes; and wherein the additional operator sequences of the polynucleotides coupled to each additional microcantilever are different.

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