Incoherent merger network for robust ratiometric gene expression response.

A ratiometric response gives an output that is proportional to the ratio between the magnitudes of two inputs. Ratio computation has been observed in nature and is also needed in the development of smart probiotics and organoids. Here, we achieve ratiometric gene expression response in bacteria Escherichia coli with the incoherent merger network. In this network, one input molecule activates expression of the output protein while the other molecule activates an intermediate protein that enhances the output's degradation. When degradation rate is first order and faster than dilution, the output responds linearly to the ratio between the input molecules' levels over a wide range with R2 close to 1. Response sensitivity can be quantitatively tuned by varying the output's translation rate. Furthermore, ratiometric responses are robust to global perturbations in cellular components that influence gene expression because such perturbations affect the output through an incoherent feedforward loop. This work demonstrates a new molecular signal processing mechanism for multiplexed sense-and-respond circuits that are robust to intra-cellular context.

Programming Fluorogenic DNA Probes for Rapid Detection of Steroids.

The ability of aptamers to recognize a variety of different molecules has fueled their emergence as recognition agents to probe complex media and cells. Many detection strategies require aptamer binding to its target to result in a dramatic change in structure, typically from an unfolded to a folded state. Here, we report a strategy based on forced intercalation (FIT) that increases the scope of aptamer recognition by transducing subtle changes in aptamer structures into fluorescent readouts. By screening a library of green-fluorescent FIT-aptamers whose design is guided by computational modeling, we could identify hits that sense steroids like dehydroepiandrosterone sulfate (DHEAS) down to 1.3 µM with no loss in binding affinity compared to the unmodified aptamer. This enabled us to study DHEAS in clinical serum samples with several advantages over gold standard methods, including rapid readout (<30 min), simple instrumentation (plate-reader), and low sample volumes (10 µL).

Physicochemical Trapping of Neurotransmitters in Polymer-Mediated Gold Nanoparticle Aggregates for Surface-Enhanced Raman Spectroscopy.

Because of the sharp distance dependence of surface-enhanced Raman spectroscopy (SERS), analyte molecules that do not exhibit strong affinity for Au/Ag often elude detection. New methods of integrating such analytes with SERS substrates are required to circumvent this limitation and expand the sensitivity of SERS to new molecules and applications. We communicate here a solution-phase, capture agent-free method of aggregating Au nanospheres in the presence of five neurotransmitters (dopamine, epinephrine, norepinephrine, serotonin, and histamine) and preventing sedimentation by encapsulating the aggregated nanospheres with polyvinylpyrrolidone, thereby trapping the neurotransmitters in close proximity to the Au nanospheres and enabling SER detection. The primary advantages of this physicochemical trapping method, which is generalizable to analytes beyond the scope of this work, are the high signal-to-noise ratio and spectral consistency down to nM levels. Normal Raman spectra and density functional theory calculations corroborate the accuracy of the spectra. Spectra collected over a wide range of concentrations were used to construct adsorption isotherms for all five neurotransmitters, from which adsorption dissociation constants were calculated, spanning from 5.7 × 10-4 M to 1.7 × 10-10 M. We expect this method to produce high quality SER spectra of any molecule with an Au affinity known or expected (based on functional groups) to be within that range. Our results have implications for plasmonic detection of these neurotransmitters, particularly for mixtures of those that exhibited disparate Au affinity in our study. We also present evidence that this method produces spectra of sufficient resolution to explore hypotheses related to surface adsorption behavior.

Reconfigurable Carbon Nanotube Multiplexed Sensing Devices.

Here we report on the fabrication of reconfigurable and solution processable nanoscale biosensors with multisensing capability, based on single-walled carbon nanotubes (SWCNTs). Distinct DNA-wrapped (hence water-soluble) CNTs were immobilized from solution onto different prepatterned electrodes on the same chip, via a low-cost dielectrophoresis (DEP) methodology. The CNTs were functionalized with specific, and different, aptamer sequences that were employed as selective recognition elements for biomarkers indicative of stress and neuro-trauma conditions. Multiplexed detection of three different biomarkers was successfully performed, and real-time detection was achieved in serum down to physiologically relevant concentrations of 50 nM, 10 nM, and 500 pM for cortisol, dehydroepiandrosterone-sulfate (DHEAS), and neuropeptide Y (NPY), respectively. Additionally, the fabricated nanoscale devices were shown to be reconfigurable and reusable via a simple cleaning procedure. The general applicability of the strategy presented, and the facile device fabrication from aqueous solution, hold great potential for the development of the next generation of low power consumption portable diagnostic assays for the simultaneous monitoring of different health parameters.

Structured DNA Aptamer Interactions with Gold Nanoparticles.

DNA aptamers that bind biomolecular targets are of interest as the recognition element in colorimetric sensors based on gold nanoparticles (AuNP), where sensor functionality is related to changes in AuNP colloidal stability upon target binding. In order to understand the role of target binding on DNA-AuNP colloidal stability, we have used high-resolution NMR to characterize the interactions of the 36 nucleotide cocaine-binding aptamer (MN4) and related aptamers with AuNPs, cocaine, and cocaine metabolites. Changes in the aptamer imino proton NMR spectra with low (20 nM) concentrations of AuNP show that the aptamers undergo fast-exchange adsorption on the nanoparticle surface. An analysis of the spectral changes and the comparison with modified MN4 aptamers shows that the AuNP binding domain is localized on stem two of the three-stemmed aptamer. The identification of an AuNP recognition domain allows for the incorporation of AuNP binding functionality into a wide variety of aptamers. AuNP-induced spectral changes are not observed for the aptamer-AuNP mixtures in the presence of cocaine, demonstrating that aptamer absorption on the AuNP surface is modulated by aptamer-target interactions. The data also show that the DNA-AuNP interactions and sensor functionality are critically dependent on aptamer folding.

Fast and Selective Plasmonic Serotonin Detection with Aptamer-Gold Nanoparticle Conjugates.

Neurotransmitters detection is critical to understanding communication between the brain and peripheral tissue. Serotonin is a key neurotransmitter linked to a number of conditions, but a full understanding of its role in disease is still lacking. The development of fast and selective serotonin detection platforms will provide researchers with tools to monitor serotonin in individuals before and after treatment for the condition of interest. Aptamer-gold nanoparticles conjugates that responded colorimetrically to serotonin with minimal response to its metabolite and other neurotransmitters were designed by simply adsorbing the DNA on the surface of AuNPs. A plasmonic assay for serotonin detection was designed with a response to biologically relevant serotonin levels. Importantly, the assay performance was not compromised when tested in filtered spiked fetal bovine serum as a mimic of biofluids. This work shows that these simple and stable Apt-AuNP conjugates are promising tools to develop fast assays for point-of-care and personalized diagnostics applications.

Cross-Reactive Plasmonic Aptasensors for Controlled Substance Identification.

In this work, we developed an assay to determine if an arbitrary white powder is a controlled substance, given the plasmonic response of aptamer-gold nanoparticle conjugates (Apt-AuNPs). Toward this end, we designed Apt-AuNPs with specific a response to common controlled substances without cross reactivity to chemicals typically used as fillers in street formulations. Plasmonic sensor variation was shown to produce unique data fingerprints for each chemical analyzed, supporting the application of multivariate statistical techniques to annotate unknown samples by chemical similarity. Importantly, the assay takes less than fifteen minutes to run, and requires only a few micrograms of the material, making the proposed assay easily deployable in field operations.

Electrokinetic preconcentration and detection of neuropeptides at patterned graphene-modified electrodes in a nanochannel.

Neuropeptides are vital to the transmission and modulation of neurological signals, with Neuropeptide Y (NPY) and Orexin A (OXA) offering diagnostic information on stress, depression, and neurotrauma. NPY is an especially significant biomarker, since it can be noninvasively collected from sweat, but its detection has been limited by poor sensitivity, long assay times, and the inability to scale-down sample volumes. Herein, we apply electrokinetic preconcentration of the neuropeptide onto patterned graphene-modified electrodes in a nanochannel by frequency-selective dielectrophoresis for 10 s or by electrochemical adsorptive accumulation for 300 s, to enable the electrochemical detection of NPY and OXA at picomolar levels from subnanoliter samples, with sufficient signal sensitivity to avoid interferences from high levels of dopamine and ascorbic acid within biological matrices. Given the high sensitivity of the methodology within small volume samples, we envision its utility toward off-line detection from droplets collected by microdialysis for the eventual measurement of neuropeptides at high spatial and temporal resolutions.

Plasmonic aptamer-gold nanoparticle sensors for small molecule fingerprint identification.

The utilization of the plasmonic response of aptamer-gold nanoparticle conjugates (Apt-AuNPs) to design cross-reactive arrays for fingerprint identification of small molecular targets was demonstrated for the first time. Four aptamers with different structural features previously selected to bind different targets were used in combination with AuNPs by adsorbing the DNA on the AuNPs surface. The optimized response of the Apt-AuNPs to the analytes showed that, depending on the specific aptamer used, target binding by the aptamer could result in an increase or decrease of Apt-AuNPs stability. These Apt-AuNPs showed the ability to recognize different analytes with different affinities, generating fingerprints that allowed unambiguous analyte identification with response times in less than fifteen minutes. Importantly, it was observed that it was not necessary to select an aptamer per analyte of interest to generate differentiable signatures, but a subset of aptamers could be used to identify a larger number of analytes. The data was analyzed using principal component analysis, showing efficient clustering of the different datasets for qualitative and quantitative identification. This work opens the door to using these Apt-AuNPs in point of care diagnostics applications where fast sensors with easy to read outputs are needed.

Tunable stringency aptamer selection and gold nanoparticle assay for detection of cortisol.

The first-known aptamer for the stress biomarker cortisol was selected using a tunable stringency magnetic bead selection strategy. The capture DNA probe immobilized on the beads was systematically lengthened to increase the number of bases bound to the complementary pool primer regions following selection enrichment. This resulted in a single sequence (15-1) dominating the final round 15 pool, where the same sequence was the second-highest copy number candidate in the enriched pool with the shorter capture DNA probe (round 13). A thorough analysis of the next-generation sequencing results showed that a high copy number may only correlate with enhanced affinity under certain stringency and enrichment conditions, in contrast with prior published reports. Aptamer 15-1 demonstrated enhanced binding to cortisol (K(d) = 6.9 ± 2.8 µM by equilibrium dialysis; 16.1 ± 0.6 µM by microscale thermophoresis) when compared with the top sequence from round 13 and the negative control progesterone. Whereas most aptamer selections terminate at the selection round demonstrating the highest enrichment, this work shows that extending the selection with higher stringency conditions leads to lower amounts eluted by the target but higher copy numbers of a sequence with enhanced binding. The structure-switching aptamer was applied to a gold nanoparticle assay in buffer and was shown to discriminate between cortisol and two other stress biomarkers, norepinephrine and epinephrine, and a structurally analogous biomarker of liver dysfunction, cholic acid. We believe this approach enhances aptamer selection and serves as proof-of-principle work toward development of point-of-care diagnostics for medical, combat, or bioterrorism targets.

Redox cycling-based signal amplification at alkanethiol modified nanoporous gold interdigitated microelectrodes.

Interdigitated electrodes (IDEs) enable electrochemical signal enhancement through repeated reduction and oxidation of the analyte molecule. Porosity on these electrodes is often used to lower the impedance background. However, their high capacitive current and signal interferences with oxygen reduction limit electrochemical detection ability. We present utilization of alkanethiol modification on nanoporous gold (NPG) electrodes to lower their background capacitance and chemically passivate them from interferences due to oxygen reduction, while maintaining their fast electron transfer rates, as validated by lower separation between anodic and cathodic peaks (ΔE) and lower charge transfer resistance (Rct) values in comparison to planar gold electrodes. Redox amplification based on this modification enables sensitive detection of various small molecules, including pyocyanin, p-aminophenol, and selective detection of dopamine in the presence of ascorbic acid. Alkanethiol NPG arrays are applied as a multiplexed sensor testbed within a well plate to screen binding of various peptide receptors to the SARS COV2 S-protein by using a sandwich assay for conversion of PAPP (4-aminophenyl phosphate) to PAP (p-aminophenol), by the action of AP (alkaline phosphatase), which is validated against optical ELISA screens of the peptides. Such arrays are especially of interest in small volume analytical settings with complex samples, wherein optical methods are unsuitable.

Rational Approach to Optimizing Conformation-Switching Aptamers for Biosensing Applications.

The utilization of structure-switching aptamers (SSAs) has enabled the development of novel sensing platforms for the sensitive and continuous detection of molecules. De novo development of SSAs, however, is complex and laborious. Here we describe a rational approach to SSA optimization that simultaneously improves aptamer binding affinity and introduces target-dependent conformation-switching for compatibility with real-world biosensor applications. Key structural features identified from NMR and computational modeling were used to optimize conformational switching in the presence of target, while large-scale, microarray-based mutation analysis was used to map regions of the aptamer permissive to mutation and identify combinations of mutations with stronger binding affinity. Optimizations were carried out in a relevant biofluid to ensure a seamless transition of the aptamer to a biosensing platform. Initial proof-of-concept for this approach is demonstrated with a cortisol binding aptamer but can easily be translated to other relevant aptamers. Cortisol is a hormone correlated with the stress response that has been associated with various medical conditions and is present at quantifiable levels in accessible biofluids. The ability to continuously track levels of stress in real-time via cortisol monitoring, which can be enabled by the aptamers reported here, is crucial for assessing human health and performance.

Highly multiplexed design of an allosteric transcription factor to sense novel ligands.

Allosteric transcription factors (aTF), widely used as biosensors, have proven challenging to design for detecting novel molecules because mutation of ligand-binding residues often disrupts allostery. We developed Sensor-seq, a high-throughput platform to design and identify aTF biosensors that bind to non-native ligands. We screened a library of 17,737 variants of the aTF TtgR, a regulator of a multidrug exporter, against six non-native ligands of diverse chemical structures - four derivatives of the cancer therapeutic tamoxifen, the antimalarial drug quinine, and the opiate analog naltrexone - as well as two native flavonoid ligands, naringenin and phloretin. Sensor-seq identified novel biosensors for each of these ligands with high dynamic range and diverse specificity profiles. The structure of a naltrexone-bound design showed shape-complementary methionine-aromatic interactions driving ligand specificity. To demonstrate practical utility, we developed cell-free detection systems for naltrexone and quinine. Sensor-seq enables rapid, scalable design of new biosensors, overcoming constraints of natural biosensors.

Sugar nucleotide quantification using multiple reaction monitoring liquid chromatography/tandem mass spectrometry.

RATIONALE: Glycosylation of proteins and lipids is reliant on the availability of monosaccharide-activated donors known as sugar nucleotides. They are responsible for glycosylation in cells. Reliable quantification of these sugar nucleotides might provide an insight into their biological roles and attributes. METHODS: Herein, a method is described for the quantification of sugar nucleotides using ultra high pressure liquid chromatography (UHPLC) tandem mass spectrometry, allowing selective detection of sugar nucleotides in a biological sample. Seven model sugar nucleotide standards commonly associated with lipid and protein glycosylation were separated on a porous graphitic carbon column using an UHPLC system coupled to a triple stage quadrupole mass spectrometer utilizing a multiple reaction monitoring approach. RESULTS: Successful baseline separation of these metabolites was attained in 6 min using an ammonium formate buffer and acetonitrile, circumventing the use of MS-unfriendly pairing reagents. The linear dynamic range of this procedure was established over almost three orders of magnitude from 20 pg to 1 ng (40 pg to 2 ng for the isomers UDP-GlcNAc/GalNAc). The limit of detection ranged from 15 pg to 30 pg while the limit of quantification ranged from 50 pg to 100 pg. Furthermore, viability of this method was tested using three different breast cancer cell lines (MDA-MB-231, MDA-MB-231-BR, and MDA-MB-361) with the successful identification and quantification of all seven targeted sugar nucleotides. CONCLUSIONS: The described method permitted the quantitative analysis of sugar nucleotides in 10 min, thus allowing the practical use of this approach in high-throughput settings. The method was also very effective for the quantification of sugar nucleotides derived from three different breast cancer cell lines. The distribution of sugar nucleotides was different among the different cell lines and unique for each cell line.

Detection of Bile Acids in Complex Matrices Using a Transcription Factor-Based Biosensor.

Bile acids play an important role in digestion and human health, are found throughout the gastrointestinal tract, and are excreted in feces. Therefore, bile acids are promising biomarkers for monitoring health and detecting fecal contamination in water sources. Here, we engineered a bile acid sensor by expressing the transcription factor BreR, a TetR-like repressor from Vibrio cholorae, in Escherichia coli. The sensor was further optimized by screening a promoter library. To further characterize the BreR sensor and increase its utility, we moved expression to a cell-free expression (CFE) system, resulting in an approximately 3 orders of magnitude increase in deoxycholic acid sensitivity. We next optimized this sensor to detect bile acids in fecal water, wastewater, and serum and transferred the CFE sensor to a paper-based assay to enhance fieldability.

Engineering a Synthetic Dopamine-Responsive Riboswitch for In Vitro Biosensing.

The detection of chemicals using natural allosteric transcription factors is a powerful strategy for point-of-use molecular sensing, particularly using fieldable cell-free gene expression (CFE) systems. However, the reliance of detection schemes on characterized protein-based sensors limits the number of measurable analytes. One alternative solution to this issue is to develop new sensors by generating RNA aptamers against the target analyte and then incorporating them directly into a riboswitch scaffold for ligand-inducible genetic control of a reporter protein. However, this strategy has not generated more than a handful of successful portable cell-free molecular sensors. To address this gap, here we convert dopamine-binding aptamers into functional dopamine-sensing riboswitches that regulate gene expression in a freeze-dried CFE reaction. We then develop an assay for direct detection and semi-quantification of dopamine in human urine. We anticipate that this work will be broadly applicable for converting many in vitro-generated RNA aptamers into fieldable molecular diagnostics.

Biofunctionalized zinc oxide field effect transistors for selective sensing of riboflavin with current modulation.

Zinc oxide field effect transistors (ZnO-FET), covalently functionalized with single stranded DNA aptamers, provide a highly selective platform for label-free small molecule sensing. The nanostructured surface morphology of ZnO provides high sensitivity and room temperature deposition allows for a wide array of substrate types. Herein we demonstrate the selective detection of riboflavin down to the pM level in aqueous solution using the negative electrical current response of the ZnO-FET by covalently attaching a riboflavin binding aptamer to the surface. The response of the biofunctionalized ZnO-FET was tuned by attaching a redox tag (ferrocene) to the 3' terminus of the aptamer, resulting in positive current modulation upon exposure to riboflavin down to pM levels.

Preparation of Multifunctional Silk-Based Microcapsules loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches.

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical cores. Following adsorption of a prime layer and DNA plasmids, the formation of robust microcapsules was facilitated by inducing ß-sheets in silk secondary structure during acute dehydration of a single silk layer. Hence, the layering occurred via multiple hydrogen bonding and hydrophobic interactions. Upon adsorption of multilayered shells, the core-shell structures can be further functionalized with gold nanoparticles (AuNPs) and/or antibodies (IgG) to be used for remote sensing and/or targeted delivery. Adjusting several key parameters during sequential deposition of key macromolecules on silica cores such as the presence of a polymer primer, the concentration of DNA and silk protein, as well as a number of adsorbed layers resulted in biocompatible, DNA-laden microcapsules with variable permeability and DNA loadings. Upon dissolution of silica cores, the protocol demonstrated the formation of hollow and robust microcapsules with DNA plasmids immobilized to the inner surface of the capsule membrane. Creating a selectively permeable biocompatible membrane between the DNA plasmids and the external environment preserved the DNA during long-term storage and played an important role in the improved output response from spatially confined plasmids. The activity of DNA templates and their accessibility were tested during in vitro transcription and translation reactions (cell-free systems). DNA plasmids encoding RNA light-up aptamers and riboswitches were successfully activated with corresponding analytes, as was visualized during localization of fluorescently labeled RNA transcripts or GFPa1 protein in the shell membranes.

Optimization of Heavy Metal Sensors Based on Transcription Factors and Cell-Free Expression Systems.

Many bacterial mechanisms for highly specific and sensitive detection of heavy metals and other hazards have been reengineered to serve as sensors. In some cases, these sensors have been implemented in cell-free expression systems, enabling easier design optimization and deployment in low-resource settings through lyophilization. Here, we apply the advantages of cell-free expression systems to optimize sensors based on three separate bacterial response mechanisms for arsenic, cadmium, and mercury. We achieved detection limits below the World Health Organization-recommended levels for arsenic and mercury and below the short-term US Military Exposure Guideline levels for all three. The optimization of each sensor was approached differently, leading to observations useful for the development of future sensors: (1) there can be a strong dependence of specificity on the particular cell-free expression system used, (2) tuning of relative concentrations of the sensing and reporter elements improves sensitivity, and (3) sensor performance can vary significantly with linear vs plasmid DNA. In addition, we show that simply combining DNA for the three sensors into a single reaction enables detection of each target heavy metal without any further optimization. This combined approach could lead to sensors that detect a range of hazards at once, such as a panel of water contaminants or all known variants of a target virus. For low-resource settings, such "all-hazard" sensors in a cheap, easy-to-use format could have high utility.

A study of the compartmentalization of core-shell nanoparticles through fluorescence energy transfer of dopants.

Hybrid organic-inorganic templates and core-shell nanoparticles were used as models to study the communication between fluorescent probes placed inside nanoparticles. The hybrid templates were prepared on the basis of a mixed-surfactant system using octadecyltrimethoxysilane as a reactive amphiphile. The core-shell particles were obtained after coating of the templates with a siloxane shell, using the silanol groups on their surface. Atomic force microscopy imaging showed that the templates were made of a flexible material that flattened significantly after deposition on a substrate and evaporation of the solvent. Pyrene was sequestered by the templates in an aqueous suspension, which placed it in a nonpolar environment, as observed by its fluorescence response. Subsequently, double-doped templates were prepared by sequestering coumarin 153 (C153), with pyrene-doped hybrid templates. The communication between these probes was studied on the basis of their spectral properties, by means of fluorescence resonance energy transfer (FRET). Energy transfer between the dyes with efficiencies up to 55% was observed. Similarly, double-doped core-shell particles prepared on the basis of the hybrid templates were doped with this pair of dyes. Despite the presence of the shell, which was intended to increment the average separation between the probes, interaction of the dyes was observed, although with lower efficiencies. A similar study was performed with C153 and 4-(dicyanomethylene)-2-methyl-6-p-(dimethylamino)styryl-4H-pyran (DCM). FRET studies indicated that the probes were placed in proximity to each other. We confirmed these observations by means of fluorescence lifetime measurements, which showed a decrease in the lifetime of the donor upon addition of the acceptor.