A near-infrared optical nanosensor for measuring aerobic respiration in microbial systems.

We developed a ratiometric oxygen-sensitive nanosensor and demonstrated application in monitoring metabolic oxygen consumption in microbial samples over time. Based on a near-infrared (NIR) emitting oxygen-quenched luminophore, platinum(II) octaethylporphine ketone (PtOEPK), along with a stable dioctadecyl dicarbocyanine reference dye (DiD), this nanosensor system provides an advantageous approach for overcoming imaging issues in biological systems, such as autofluorescence and optical scattering in the visible wavelength region. The dyes are encapsulated within a polymer-based nanoparticle matrix to maintain them at a constant ratio in biological samples, precluding the need for complex synthetic approaches. With this constant ratio of the two dyes, the nanosensor response can be measured as a ratio of their two signals, accounting for nanosensor concentration artifacts in measurements. The nanosensors are reversible, which enabled us to temporally monitor systems in which dissolved oxygen concentrations both increase and decrease. These sensors were applied for the monitoring of oxygen in samples of Saccharomyces cerevisiae (brewing yeast) in a 96-well optical fluorescence plate reader format over 60 h. By mixing the nanosensors directly into the sample well with the yeast, we were able to dynamically track metabolic activity changes over time due to varying cell concentration and exposure to an antimicrobial agent. This system could be a potential platform for high-throughput screening of various species or variants of microbes with unknown metabolic rates in response to external stimuli (antimicrobials, metabolites, etc.).

Nanodiagnostics to monitor biofilm oxygen metabolism for antibiotic susceptibility testing.

In clinical environments, many serious antibiotic-resistant infections are caused by biofilm-forming species. This presents issues when attempting to determine antimicrobial dosing as traditional antibiotic susceptibility tests (ASTs) are typically designed around planktonic bacteria and thus offer information that is not relevant to the biofilm phenotype present in the patient. Even the popular Calgary biofilm device may provide inaccurate minimum biofilm inhibitory concentrations (MBICs) and can be time- and material-intensive. In this work, we present a method utilizing oxygen-sensitive nanosensor technology to monitor the oxygen consumption dynamics of living biofilms as they are exposed to antibiotics. We incorporated our nanosensors into biofilms grown from P. aeruginosa strains of varying sensitivity to traditional classes of antibiotics. Through measuring nanosensor response under antibiotic administration we determined the concentrations able to cease biofilm metabolism. This method provides information on the MBIC as well as kinetic response information in a manner that requires fewer materials and is more reflective of biofilm behavior than a traditional AST.

Luminescent Nanosensors for Ratiometric Monitoring of Three-Dimensional Oxygen Gradients in Laboratory and Clinical Pseudomonas aeruginosa Biofilms.

Bacterial biofilms can form persistent infections on wounds and implanted medical devices and are associated with many chronic diseases, such as cystic fibrosis. These infections are medically difficult to treat, as biofilms are more resistant to antibiotic attack than their planktonic counterparts. An understanding of the spatial and temporal variation in the metabolism of biofilms is a critical component toward improved biofilm treatments. To this end, we developed oxygen-sensitive luminescent nanosensors to measure three-dimensional (3D) oxygen gradients, an application of which is demonstrated here with Pseudomonas aeruginosa biofilms. The method was applied here and improves on traditional one-dimensional (1D) methods of measuring oxygen profiles by investigating the spatial and temporal variation of oxygen concentration when biofilms are challenged with antibiotic attack. We observed an increased oxygenation of biofilms that was consistent with cell death from comparisons with antibiotic kill curves for PAO1. Due to the spatial and temporal nature of our approach, we also identified spatial and temporal inhomogeneities in the biofilm metabolism that are consistent with previous observations. Clinical strains of P. aeruginosa subjected to similar interrogation showed variations in resistance to colistin and tobramycin, which are two antibiotics commonly used to treat P. aeruginosa infections in cystic fibrosis patients.IMPORTANCE Biofilm infections are more difficult to treat than planktonic infections for a variety of reasons, such as decreased antibiotic penetration. Their complex structure makes biofilms challenging to study without disruption. To address this limitation, we developed and demonstrated oxygen-sensitive luminescent nanosensors that can be incorporated into biofilms for studying oxygen penetration, distribution, and antibiotic efficacy-demonstrated here with our sensors monitoring antibiotic impacts on metabolism in biofilms formed from clinical isolates. The significance of our research is in demonstrating not only a nondisruptive method for imaging and measuring oxygen in biofilms but also that this nanoparticle-based sensing platform can be modified to measure many different ions and small molecule analytes.

Fluorescent sensors for the basic metabolic panel enable measurement with a smart phone device over the physiological range.

The advanced functionality of portable devices such as smart phones provides the necessary hardware to potentially perform complex diagnostic measurements in any setting. Recent research and development have utilized cameras and data acquisition properties of smart phones to create diagnostic approaches for a variety of diseases or pollutants. However, in concentration measurements, such as blood glucose, the performance of handheld diagnostic devices depends largely on the sensing mechanism. To expand measurements to multiple components, often necessary in medical tests, with a single diagnostic device, robust platform based sensors are needed. Here, we developed a suite of dual wavelength fluorescent sensors with response characteristics necessary to measure each component of a basic metabolic panel, a common clinical measurement. Furthermore, the response of these sensors could be measured with a simple optical setup to convert a smart phone into a fluorescence measurement instrument. This approach could be used as a mobile basic metabolic panel measurement system for point of care diagnostics.

Mini Winnies: scaled down and transparent Winogradsky columns for microscopy in microbiology education.

Winogradsky columns were invented by Sergei Winogradsky in the 1880s and have commonly been used as a microbiology classroom learning tool in K-12 and collegiate education. However, they can be challenging to examine with microscopy. We scaled down Winogradsky columns into nuclear magnetic resonance (NMR) tubes and replaced the natural sediment with a transparent soil substitute toward the goal of observing the microbial growth under a bright-field microscope without column disassembly. Using this "Mini Winnie" approach, students can practice their microscopy skills while observing microbial growth inside the column after only days of incubation on the laboratory windowsill. Overall, we believe that the Mini Winnies provide a simple method for maximizing student engagement while giving them a greater understanding of how microorganisms interact in the environment.

Persistent Luminescence Nanosensors: A Generalized Optode-Based Platform for Autofluorescence-Free Sensing in Biological Systems.

Fluorescent nanosensors have revolutionized diagnostics and our ability to monitor cellular dynamics. Yet, distinguishing sensor signals from autofluorescence remains a challenge. Here, we merged optode-based sensing with near-infrared-emitting ZnGa2O4:Cr3+ persistent luminescence nanoparticles (PLNPs) to create nanocomposites for autofluorescence-free "glow-in-the-dark" sensing. Hydrophobic modification and incorporation of the persistent luminescence nanoparticles into an optode-based nanoparticle core yielded persistent luminescence nanosensors (PLNs) for five analytes (K+, Na+, Ca2+, pH, and O2) via two distinct mechanisms. We demonstrated the viability of the PLNs by quantifying K+ in fetal bovine serum, calibrating the pH PLNs in the same, and ratiometrically monitoring O2 metabolism in cultures of Saccharomyces cerevisiae, all the while overcoming their respective autofluorescence signatures. This highly modular platform allows for facile tuning of the sensing functionality, optical properties, and surface chemistry and promises high signal-to-noise ratios in complex optical environments.

Increasing aggregate size reduces single-cell organic carbon incorporation by hydrogel-embedded wetland microbes.

Microbial degradation of organic carbon in sediments is impacted by the availability of oxygen and substrates for growth. To better understand how particle size and redox zonation impact microbial organic carbon incorporation, techniques that maintain spatial information are necessary to quantify elemental cycling at the microscale. In this study, we produced hydrogel microspheres of various diameters (100, 250, and 500 µm) and inoculated them with an aerobic heterotrophic bacterium isolated from a freshwater wetland (Flavobacterium sp.), and in a second experiment with a microbial community from an urban lacustrine wetland. The hydrogel-embedded microbial populations were incubated with 13C-labeled substrates to quantify organic carbon incorporation into biomass via nanoSIMS. Additionally, luminescent nanosensors enabled spatially explicit measurements of oxygen concentrations inside the microspheres. The experimental data were then incorporated into a reactive-transport model to project long-term steady-state conditions. Smaller (100 µm) particles exhibited the highest microbial cell-specific growth per volume, but also showed higher absolute activity near the surface compared to the larger particles (250 and 500 µm). The experimental results and computational models demonstrate that organic carbon availability was not high enough to allow steep oxygen gradients and as a result, all particle sizes remained well-oxygenated. Our study provides a foundational framework for future studies investigating spatially dependent microbial activity in aggregates using isotopically labeled substrates to quantify growth.

Multifunction fluorescence open source in vivo/in vitro imaging system (openIVIS).

The widespread availability and diversity of open-source microcontrollers paired with off-the-shelf electronics and 3D printed technology has led to the creation of a wide range of low-cost scientific instruments, including microscopes, spectrometers, sensors, data loggers, and other tools that can be used for research, education, and experimentation. These devices can be used to explore a wide range of scientific topics, from biology and chemistry to physics and engineering. In this study, we designed and built a multifunction fluorescent open source in vivo/in vitro imaging system (openIVIS) system that integrates a Raspberry Pi with commercial cameras and LEDs with 3D printed structures combined with an acrylic housing. Our openIVIS provides three excitation wavelengths of 460 nm, 520 nm, and 630 nm integrated with Python control software to enable fluorescent measurements across the full visible light spectrum. To demonstrate the potential applications of our system, we tested its performance against a diverse set of experiments including laboratory assays (measuring fluorescent dyes, using optical nanosensors, and DNA gel electrophoresis) to potentially fieldable applications (plant and mineral imaging). We also tested the potential use for a high school biology environment by imaging small animals and tracking their development over the course of ten days. Our system demonstrated its ability to measure a wide dynamic range fluorescent response from millimolar to picomolar concentrations in the same sample while measuring responses across visible wavelengths. These results demonstrate the power and flexibility of open-source hardware and software and how it can be integrated with customizable manufacturing to create low-cost scientific instruments with a wide range of applications. Our study provides a promising model for the development of low-cost instruments that can be used in both research and education.

Phosphorescent nanosensors for in vivo tracking of histamine levels.

Continuously tracking bioanalytes in vivo will enable clinicians and researchers to profile normal physiology and monitor diseased states. Current in vivo monitoring system designs are limited by invasive implantation procedures and biofouling, limiting the utility of these tools for obtaining physiologic data. In this work, we demonstrate the first success in optically tracking histamine levels in vivo using a modular, injectable sensing platform based on diamine oxidase and a phosphorescent oxygen nanosensor. Our new approach increases the range of measurable analytes by combining an enzymatic recognition element with a reversible nanosensor capable of measuring the effects of enzymatic activity. We use these enzyme nanosensors (EnzNS) to monitor the in vivo histamine dynamics as the concentration rapidly increases and decreases due to administration and clearance. The EnzNS system measured kinetics that match those reported from ex vivo measurements. This work establishes a modular approach to in vivo nanosensor design for measuring a broad range of potential target analytes. Simply replacing the recognition enzyme, or both the enzyme and nanosensor, can produce a new sensor system capable of measuring a wide range of specific analytical targets in vivo.

Triplet-Triplet Annihilation Upconversion-Based Oxygen Sensors to Overcome the Limitation of Autofluorescence.

Autofluorescence is one of the many challenges in bioimaging as it can mask the emission from fluorescent probes or markers, a limitation that can be overcome via upconversion. Herein, we have developed a nanosensor that uses triplet-triplet annihilation upconversion to optically report changes in the dissolved oxygen concentration. Using a sensitizer-annihilator dye pairing of platinum(II) octaethylporphyrin and 9,10-diphenylanthracene, we monitored the oxygen consumption (as a proxy for metabolic activity) over time in a biological system─Saccharomyces cerevisiae (brewing yeast). The nanosensor demonstrated good reversibility over multiple cycles and showed good signal and colloidal stability when tested over the course of 7 days, and it was sensitive to dissolved oxygen from 0.00 to 3.17 mg/L O2. Additionally, there was no signal overlap between the nanosensor emission and S. cerevisiae autofluorescence, thus underscoring the utility of upconversion as a facile and economical means of overcoming autofluorescence.

Wash-free, electrochemical platform for the quantitative, multiplexed detection of specific antibodies.

The diagnosis, prevention, and treatment of many illnesses, including infectious and autoimmune diseases, would benefit from the ability to measure specific antibodies directly at the point of care. Thus motivated, we designed a wash-free, electrochemical method for the rapid, quantitative detection of specific antibodies directly in undiluted, unprocessed blood serum. Our approach employs short, contiguous polypeptide epitopes coupled to the distal end of an electrode-bound nucleic acid "scaffold" modified with a reporting methylene blue. The binding of the relevant antibody to the epitope reduces the efficiency with which the redox reporter approaches, and thus exchanges electrons with, the underlying sensor electrode, producing readily measurable change in current. To demonstrate the versatility of the approach, we fabricated a set of six such sensors, each aimed at the detection of a different monoclonal antibody. All six sensors are sensitive (subnanomolar detection limits), rapid (equilibration time constants ∼8 min), and specific (no appreciable cross reactivity with the targets of the other five). When deployed in a millimeter-scale, an 18-pixel array with each of the six sensors in triplicate support the simultaneous measurement of the concentrations of multiple antibodies in a single, submilliliter sample volume. The described sensor platform thus appears be a relatively general approach to the rapid and specific quantification of antibodies in clinical materials.

In vivo histamine optical nanosensors.

In this communication we discuss the development of ionophore based nanosensors for the detection and monitoring of histamine levels in vivo. This approach is based on the use of an amine-reactive, broad spectrum ionophore which is capable of recognizing and binding to histamine. We pair this ionophore with our already established nanosensor platform, and demonstrate in vitro and in vivo monitoring of histamine levels. This approach enables capturing rapid kinetics of histamine after injection, which are more difficult to measure with standard approaches such as blood sampling, especially on small research models. The coupling together of in vivo nanosensors with ionophores such as nonactin provide a way to generate nanosensors for novel targets without the difficult process of designing and synthesizing novel ionophores.

Sensors in a Flash! Oxygen Nanosensors for Microbial Metabolic Monitoring Synthesized by Flash Nanoprecipitation.

Flash nanoprecipitation (FNP) is an efficient and scalable nanoparticle synthesis method that has not previously been applied to nanosensor fabrication. Current nanosensor fabrication methods have traditionally exhibited poor replicability and consistency resulting in high batch-to-batch variability, highlighting the need for a more tunable and efficient method such as FNP. We used FNP to fabricate nanosensors to sense oxygen based on an oxygen-sensitive dye and a reference dye, as a tool for measuring microbial metabolism. We used fluorescence spectroscopy to optimize nanosensor formulations, calibrate the nanosensors for oxygen concentration determination, and measure oxygen concentrations through oxygen-sensitive dye luminescence. FNP provides an effective platform for making sensors capable of responding to oxygen concentration in gas-bubbled solutions as well as in microbial environments. The environments we tested the sensors in arePseudomonas aeruginosa biofilms andSaccharomyces cerevisiae liquid cultures─both settings where oxygen concentration is highly dependent on microbial activity. With FNP now applied to nanosensor fabrication, future nanosensor applications can take advantage of improved product quality through better replicability and consistency while maintaining the original function of the nanosensor.

Tracking a molecular motor with a nanoscale optical encoder.

Optical encoders are commonly used in macroscopic machines to make precise measurements of distance and velocity by translating motion into a periodic signal. Here we show how Forster resonance energy transfer can be used to implement this technique at the single-molecule scale. We incorporate a series of acceptor dye molecules into self-assembling DNA, and the periodic signal resulting from unhindered motion of a donor-labeled molecular motor provides nanometer-scale resolution in milliseconds.

Nanoparticle-Based Liquid-Liquid Extraction for the Determination of Metal Ions.

Traditional liquid phase extraction techniques that use optically responsive ligands provide benefits that enable cost-efficient and rapid measurements. However, these approaches have limitations in their excessive use of organic solvents and multistep procedures. Here, we developed a simple, nanoscale extraction approach by replacing the macroscopic organic phase with hydrophobic polymeric nanoparticles that are dispersed in an aqueous feed. The concentration of analytes in polymeric nanoparticle suspensions is governed by similar partition principles to liquid-liquid phase extraction techniques. By encasing optically responsive metal ligands inside polymeric nanoparticles, we introduce a one-step metal quantification assay based on traditional two-phase extraction methodologies. As an initial proof of concept, we encapsulated bathophenanthroline (BP) inside the particles to extract then quantify Fe2+ with colorimetry in a dissolved supplement tablet and creek water. These Fe2+ nanosensors are sensitive and selective and report out with fluorescence by adding a fluorophore (DiO) into the particle core. To show that this new rapid extraction assay is not exclusive to measuring Fe2+, we replaced BP with either 8-hydroxyquinoline or bathocuproine to measure Al3+ or Cu+, respectively, in water samples. Utilizing this nanoscale extraction approach will allow users to rapidly quantify metals of interest without the drawbacks of larger-scale phase extraction approaches while also allowing for the expansion of phase extraction methodologies into areas of biological research.

Triplet-Triplet Annihilation Upconversion Based Nanosensors for Fluorescence Detection of Potassium.

Typical ionophore-based nanosensors use Nile blue derived indicators called chromoionophores, which must contend with strong background absorption, autofluorescence, and scattering in biological samples that limit their usefulness. Here, we demonstrate potassium-selective nanosensors that utilize triplet-triplet annihilation upconversion to minimize potential optical interference in biological media and a pH-sensitive quencher molecule to modulate the upconversion intensity in response to changes in analyte concentration. A triplet-triplet annihilation dye pair (platinum(II) octaethylporphyrin and 9,10-diphenylanthracene) was integrated into nanosensors containing an analyte binding ligand (ionophore), charge-balancing additive, and a pH indicator quencher. The nanosensor response to potassium was shown to be reversible and stable for 3 days. In addition, the nanosensors are selective against sodium, calcium, and magnesium (selectivity coefficients in log10 units of -2.2 for calcium, -2.0 for sodium, and -2.4 for magnesium), three interfering ions found in biological samples. The lack of signal overlap between the upconversion nanosensors and GFP, a common biological fluorescent indicator, is demonstrated in confocal microscope images of sensors embedded in a bacterial biofilm.

LipiSensors: Exploiting Lipid Nanoemulsions to Fabricate Ionophore-Based Nanosensors.

Ionophore-based nanosensors (IBNS) are tools that enable quantification of analytes in complex chemical and biological systems. IBNS methodology is adopted from that of bulk optodes where an ion exchange event is converted to a change in optical output. While valuable, an important aspect for application is the ability to intentionally tune their size with simple approaches, and ensure that they contain compounds safe for application. Lipidots are a platform of size tunable lipid nanoemulsions with a hydrophobic lipid core typically used for imaging and drug delivery. Here, we present LipiSensors as size tunable IBNS by exploiting the Lipidot model as a hydrophobic structural support for the sensing moieties that are traditionally encased in plasticized PVC nanoparticles. The LipiSensors we demonstrate here are sensitive and selective for calcium, reversible, and have a lifetime of approximately one week. By changing the calcium sensing components inside the hydrophobic core of the LipiSensors to those sensitive for oxygen, they are also able to be used as ratiometric O2 sensitive nanosensors via a quenching-based mechanism. LipiSensors provide a versatile, general platform nanosensing with the ability to directly tune the size of the sensors while including biocompatible materials as the structural support by merging sensing approaches with the Lipidot platform.

The length and viscosity dependence of end-to-end collision rates in single-stranded DNA.

Intramolecular dynamics play an essential role in the folding and function of biomolecules and, increasingly, in the operation of many biomimetic technologies. Thus motivated we have employed both experiment and simulation to characterize the end-to-end collision dynamics of unstructured, single-stranded DNAs ranging from 6 to 26 bases. We find that, because of the size and flexibility of the optical reporters employed experimentally, end-to-end collision dynamics exhibit little length dependence at length scales <11 bases. For longer constructs, however, the end-to-end collision rate exhibits a power-law relationship to polymer length with an exponent of -3.49 +/- 0.13. This represents a significantly stronger length dependence than observed experimentally for unstructured polypeptides or predicted by polymer scaling arguments. Simulations indicate, however, that the larger exponent stems from electrostatic effects that become important over the rather short length scale of these highly charged polymers. Finally, we have found that the end-to-end collision rate also depends linearly on solvent viscosity, with an experimentally significant, nonzero intercept (the extrapolated rate at zero viscosity) that is independent of chain length--n observation that sheds new light on the origins of the "internal friction" observed in the dynamics of many polymer systems.

An electrochemical sensor for the detection of protein-small molecule interactions directly in serum and other complex matrices.

Here we have demonstrated a general, sensitive, and selective approach for the detection of macromolecules that bind to specific small molecule recognition elements. Our electrochemical approach utilizes a redox-tagged DNA signaling scaffold that is conjugated to a small molecule recognition element and is covalently attached to an interrogating electrode. The binding of a protein to the small molecule recognition element alters the dynamics of the scaffold, increasing or decreasing the efficiency with which the redox tag collides with the electrode and thus altering the observed faradaic current. We optimized the scaffold using a biotin recognition element and streptavidin as a target to determine the variables that define sensor performance before then applying the approach to detection of anti-digoxigenin antibodies using the steroid as the recognition element. We generated streptavidin sensors exhibiting both signal-on (target binding increases the faradaic current) and signal-off behavior, of which only the signal-off approach was generalizable to the detection of antibodies. Sensors for both targets are sensitive (detection limits in the low nanomolar range), rapid (minutes), reusable, and selective enough to function directly in complex matrices including blood serum, soil, and foodstuffs.

Optimization of a reusable, DNA pseudoknot-based electrochemical sensor for sequence-specific DNA detection in blood serum.

We describe in detail a new electrochemical DNA (E-DNA) sensing platform based on target-induced conformation changes in an electrode-bound DNA pseudoknot. The pseudoknot, a DNA structure containing two stem-loops in which the first stem's loop forms part of the second stem, is modified with a methylene blue redox tag at its 3' terminus and covalently attached to a gold electrode via the 5' terminus. In the absence of a target, the structure of the pseudoknot probe minimizes collisions between the redox tag and the electrode, thus reducing faradaic current. Target binding disrupts the pseudoknot structure, liberating a flexible, single-stranded element that can strike the electrode and efficiently transfer electrons. In this article we report further characterization and optimization of this new E-DNA architecture. We find that optimal signaling is obtained at an intermediate probe density ( approximately 1.8 x 10(13) molecules/cm(2) apparent density), which presumably represents a balance between steric and electrostatic blocking at high probe densities and increased background currents arising from transfer from the pseudoknot probe at lower densities. We also find that optimal 3' stem length, which appears to be 7 base pairs, represents a balance between pseudoknot structural stability and target affinity. Finally, a 3' loop comprised of poly(A) exhibits better mismatch discrimination than the equivalent poly(T) loop, but at the cost of decreased gain. Optimization over this parameter space significantly improves the signaling of the pseudoknot-based E-DNA architecture, leading to the ability to sensitively and specifically detect DNA targets even when challenged in complex, multicomponent samples such as blood serum.