Substrate-Wrapped, Single-Walled Carbon Nanotube Probes for Hydrolytic Enzyme Characterization

Hydrolytic enzymes are a topic of continual study and improvement due to their industrial impact and biological implications; however, the ability to measure the activity of these enzymes, especially in high-throughput assays, is limited to an established, few enzymes and often involves the measurement of secondary byproducts or the design of a complex degradation probe. Herein, a versatile single-walled carbon nanotube (SWNT)-based biosensor that is straightforward to produce and measure is described. The hydrolytic enzyme substrate is rendered as an amphiphilic polymer, which is then used to solubilize the hydrophobic nanotubes. When the target enzyme degrades the wrapping, the SWNT fluorescent signal is quenched due to increased solvent accessibility and aggregation, allowing quantitative measurement of hydrolytic enzyme activity. Using (6,5) chiral SWNT suspended with polypeptides and polysaccharides, turnover frequencies are estimated for cellulase, pectinase, and bacterial protease. Responses are recorded for concentrations as low as 5 fM using a well-characterized protease, Proteinase K. An established trypsin-based plate reader assay is used to compare this nanotube probe assay with standard techniques. Furthermore, the effect of freeze–thaw cycles and elevated temperature on enzyme activity is measured, suggesting freezing to have minimal impact even after 10 cycles and heating to be detrimental above 60 °C. Finally, rapid optimization of enzyme operating conditions is demonstrated by generating a response surface of cellulase activity with respect to temperature and pH to determine optimal conditions within 2 h of serial scans

Resonant Sensors for Low-Cost, Contact-Free Measurement of Hydrolytic Enzyme Activity in Closed Systems

A passive, resonant sensor was developed that can be embedded in closed systems for wireless monitoring of hydrolytic enzyme activity. The resonators are rapidly prototyped from copper coated polyimide substrates that are masked using an indelible marker with an <i>XY</i> plotter and subsequently etched. The resonator’s frequency response window is designed by the Archimedean coil length and pitch and is tuned for the 1–100 MHz range for better penetration through soil, water, and tissue. The resonant frequency is measured up to 5 cm stand-off distance by a coplanar, two-loop coil reader antenna attached to a vector network analyzer monitoring the S21 scattering parameter. The resonant frequency is modulated (up to 50 MHz redshift) by changing the relative permittivity of the medium in contact with the resonator (e.g., air to water). The resonant sensors are coated by an enzyme substrate, which, when degraded, causes a change in dielectric and a shift in resonant frequency (up to 7 MHz redshift). The activity (turnover rate, or <i>k</i>_cat) of the enzyme is calculated by fitting the measured data via a custom transport and reaction model which simulates the radial digestion profile. This is used to test purified Subtilisin A and unpurified bacterial protease samples at concentrations of 30 mg/mL to 200 mg/mL with <i>k</i>_cat ranges of 0.003–0.002 and 0.008–0.004 <i>g</i>_substrate/<i>g</i>_enzyme per second. The sensor response rate can be tuned by substrate composition (e.g., gelatin and glycerol plasticizer weight percentage). Finally, the utility of these sensors is demonstrated by wirelessly measuring the proteolytic activity of farm soil with a measured <i>k</i>_cat of 0.00152 <i>g</i>_substrate/(<i>g</i>_soil·s)

Three-Dimensional Tracking of Carbon Nanotubes within Living Cells

Three-dimensional tracking of single-walled carbon nanotubes (SWNT) with an orbital tracking microscope is demonstrated. We determine the viscosity regime (above 250 cP) at which the rotational diffusion coefficient can be used for length estimation. We also demonstrate SWNT tracking within live HeLa cells and use these findings to spatially map corral volumes (0.27–1.32 μm³), determine an active transport velocity (455 nm/s), and calculate local viscosities (54–179 cP) within the cell. With respect to the future use of SWNTs as sensors in living cells, we conclude that the sensor must change the fluorescence signal by at least 4–13% to allow separation of the sensor signal from fluctuations due to rotation of the SWNT when measuring with a time resolution of 32 ms

Hydrolytic Enzymes as (Bio)-Logic for Wireless and Chipless Biosensors

The switchable activity of allosteric, hydrolytic enzymes was used as a single-input, “buffer” logic gate (performing YES and NOT) in a screen-printable biosensor. The enzyme substrate functioned as an “AND” logic gate with the enzyme and cofactor as inputs. These (bio)-logic materials transduced a signal by the cofactor activating the enzyme which then degraded the substrate that formed the dielectric of a tuning capacitor in an inductor-capacitor (LC) circuit. The degradation of the substrate was engineered to shift the capacitance and thus the resonant frequency of the device. The resonant frequency was monitored wirelessly with a low-power vector network analyzer observing the S21 parameter. Proof of concept was shown with subtilisin as the enzyme, activated by calcium (100 μg/mL and 5 mM, respectively) degrading a collagen substrate with a demonstrated wireless read range of up to 4 cm. Selectivity over other divalent cations (magnesium, copper II, and manganese II) and the effect of receiver motion were also shown on the wireless measurement

A Structure–Function Relationship for the Optical Modulation of Phenyl Boronic Acid-Grafted, Polyethylene Glycol-Wrapped Single-Walled Carbon Nanotubes

Phenyl boronic acids (<b>PBA</b>) are important binding ligands to pendant diols useful for saccharide recognition. The aromatic ring can also function to anchor an otherwise hydrophilic polymer backbone to the surface of hydrophobic graphene or carbon nanotube. In this work, we demonstrate both functions using a homologous series of seven phenyl boronic acids conjugated to a polyethylene glycol, eight-membered, branched polymer (<b>PPEG8</b>) that allows aqueous dispersion of single-walled carbon nanotubes (SWNT) and quenching of the near-infrared fluorescence in response to saccharide binding. We compare the 2-carboxyphenylboronic acid (<b>2CPBA</b>); 3-carboxy- (<b>3CPBA</b>) and 4-carboxy- (<b>4CPBA</b>) phenylboronic acids; <i>N</i>-(4-phenylboronic)­succinamic acid (<b>4SCPBA</b>); 5-bromo-3-carboxy- (<b>5B3CPBA</b>), 3-carboxy-5-fluoro- (<b>5F3CPBA</b>), and 3-carboxy-5-nitro- (<b>5N3CPBA</b>) phenylboronic acids, demonstrating a clear link between SWNT photoluminescence quantum yield and boronic acid structure. Surprisingly, quantum yield decreases systematically with both the location of the BA functionality and the inclusion of electron-withdrawing or -donating substituents on the phenyl ring. For three structural isomers (<b>2CPBA</b>, <b>3CPBA</b>, and <b>4CPBA</b>), the highest quantum yields were measured for para-substituted PBA (<b>4CPBA</b>), much higher than ortho- (<b>2CPBA</b>) and meta- (<b>3CPBA</b>) substituted PBA, indicating the first such dependence on molecular structure. Electron-withdrawing substituents such as nitro groups on the phenyl ring cause higher quantum yield, while electron-donating groups such as amides and alkyl groups cause a decrease. The solvatochromic shift of up to 10.3 meV was used for each case to estimate polymer surface coverage on an areal basis using a linear dielectric model. Saccharide recognition using the nIR photoluminescence of SWNT is demonstrated, including selectivity toward pentoses such as arabinose, ribose, and xylose to the exclusion of the expected fructose, which has a high selectivity on PBA due to the formation of a tridentate complex between fructose and PBA. This study is the first to conclusively link molecular structure of an adsorbed phase to SWNT optical properties and modulation in a systematic manner

Neurotransmitter Detection Using Corona Phase Molecular Recognition on Fluorescent Single-Walled Carbon Nanotube Sensors

Temporal and spatial changes in neurotransmitter concentrations are central to information processing in neural networks. Therefore, biosensors for neurotransmitters are essential tools for neuroscience. In this work, we applied a new technique, corona phase molecular recognition (CoPhMoRe), to identify adsorbed polymer phases on fluorescent single-walled carbon nanotubes (SWCNTs) that allow for the selective detection of specific neurotransmitters, including dopamine. We functionalized and suspended SWCNTs with a library of different polymers (<i>n</i> = 30) containing phospholipids, nucleic acids, and amphiphilic polymers to study how neurotransmitters modulate the resulting band gap, near-infrared (nIR) fluorescence of the SWCNT. We identified several corona phases that enable the selective detection of neurotransmitters. Catecholamines such as dopamine increased the fluorescence of specific single-stranded DNA- and RNA-wrapped SWCNTs by 58–80% upon addition of 100 μM dopamine depending on the SWCNT chirality (<i>n</i>,<i>m</i>). In solution, the limit of detection was 11 nM [<i>K</i>_d = 433 nM for (GT)₁₅ DNA-wrapped SWCNTs]. Mechanistic studies revealed that this turn-on response is due to an increase in fluorescence quantum yield and not covalent modification of the SWCNT or scavenging of reactive oxygen species. When immobilized on a surface, the fluorescence intensity of a single DNA- or RNA-wrapped SWCNT is enhanced by a factor of up to 5.39 ± 1.44, whereby fluorescence signals are reversible. Our findings indicate that certain DNA/RNA coronae act as conformational switches on SWCNTs, which reversibly modulate the SWCNT fluorescence. These findings suggest that our polymer–SWCNT constructs can act as fluorescent neurotransmitter sensors in the tissue-compatible nIR optical window, which may find applications in neuroscience

Neurotransmitter Detection Using Corona Phase Molecular Recognition on Fluorescent Single-Walled Carbon Nanotube Sensors

Temporal and spatial changes in neurotransmitter concentrations are central to information processing in neural networks. Therefore, biosensors for neurotransmitters are essential tools for neuroscience. In this work, we applied a new technique, corona phase molecular recognition (CoPhMoRe), to identify adsorbed polymer phases on fluorescent single-walled carbon nanotubes (SWCNTs) that allow for the selective detection of specific neurotransmitters, including dopamine. We functionalized and suspended SWCNTs with a library of different polymers (<i>n</i> = 30) containing phospholipids, nucleic acids, and amphiphilic polymers to study how neurotransmitters modulate the resulting band gap, near-infrared (nIR) fluorescence of the SWCNT. We identified several corona phases that enable the selective detection of neurotransmitters. Catecholamines such as dopamine increased the fluorescence of specific single-stranded DNA- and RNA-wrapped SWCNTs by 58–80% upon addition of 100 μM dopamine depending on the SWCNT chirality (<i>n</i>,<i>m</i>). In solution, the limit of detection was 11 nM [<i>K</i>_d = 433 nM for (GT)₁₅ DNA-wrapped SWCNTs]. Mechanistic studies revealed that this turn-on response is due to an increase in fluorescence quantum yield and not covalent modification of the SWCNT or scavenging of reactive oxygen species. When immobilized on a surface, the fluorescence intensity of a single DNA- or RNA-wrapped SWCNT is enhanced by a factor of up to 5.39 ± 1.44, whereby fluorescence signals are reversible. Our findings indicate that certain DNA/RNA coronae act as conformational switches on SWCNTs, which reversibly modulate the SWCNT fluorescence. These findings suggest that our polymer–SWCNT constructs can act as fluorescent neurotransmitter sensors in the tissue-compatible nIR optical window, which may find applications in neuroscience

Emergent Properties of Nanosensor Arrays: Applications for Monitoring IgG Affinity Distributions, Weakly Affined Hypermannosylation, and Colony Selection for Biomanufacturing

It is widely recognized that an array of addressable sensors can be multiplexed for the label-free detection of a library of analytes. However, such arrays have useful properties that emerge from the ensemble, even when monofunctionalized. As examples, we show that an array of nanosensors can estimate the mean and variance of the observed dissociation constant (<i>K</i>_D), using three different examples of binding IgG with Protein A as the recognition site, including polyclonal human IgG (<i>K</i>_D μ = 19 μM, σ² = 1000 mM²), murine IgG (<i>K</i>_D μ = 4.3 nM, σ² = 3 μM²), and human IgG from CHO cells (<i>K</i>_D μ = 2.5 nM, σ² = 0.01 μM²). Second, we show that an array of nanosensors can uniquely monitor weakly affined analyte interactions <i>via</i> the increased number of observed interactions. One application involves monitoring the metabolically induced hypermannosylation of human IgG from CHO using PSA-lectin conjugated sensor arrays where temporal glycosylation patterns are measured and compared. Finally, the array of sensors can also spatially map the local production of an analyte from cellular biosynthesis. As an example, we rank productivity of IgG-producing HEK colonies cultured directly on the array of nanosensors itself