Nitrogen-Vacancy Magnetic Relaxometry of Nanoclustered Cytochrome C Proteins.

Nitrogen-vacancy (NV) magnetometry offers an alternative tool to detect paramagnetic centers in cells with a favorable combination of magnetic sensitivity and spatial resolution. Here, we employ NV magnetic relaxometry to detect cytochrome C (Cyt-C) nanoclusters. Cyt-C is a water-soluble protein that plays a vital role in the electron transport chain of mitochondria. Under ambient conditions, the heme group in Cyt-C remains in the Fe3+ state, which is paramagnetic. We vary the concentration of Cyt-C from 6 to 54 µM and observe a reduction of the NV spin-lattice relaxation time (T1) from 1.2 ms to 150 µs, which is attributed to the spin noise originating from the Fe3+ spins. NV T1 imaging of Cyt-C drop-casted on a nanostructured diamond chip allows us to detect the relaxation rates from the adsorbed Fe3+ within Cyt-C.

Streamlined Formation and Manipulation of Charged Polymersomes.

Charged polymersomes are attractive for advanced material applications due to their versatile encapsulation capabilities and charge-induced functionality. Although desirable, the pH-sensitivity of charged block copolymers adds complexity to its self-assembly process, making it challenging to produce charged polymersomes in a reliable manner. In this work, a flow approach to control and strike a delicate balance between solvent composition and pH for self-assembly is used. This allows for the identification of a phase window to reliably produce of charged polymersomes. The utility of this approach to streamline downstream processes, such as morphological transformation or in-line purification is further demonstrated. As proof-of-concept, it is shown that the processed polymersomes can be used for surface modifications facilitated by charge complexation.

Multiplexed Monitoring of Neurochemicals via Electrografting-Enabled Site-Selective Functionalization of Aptamers on Field-Effect Transistors.

Neurochemical corelease has received much attention in understanding brain activity and cognition. Despite many attempts, the multiplexed monitoring of coreleased neurochemicals with spatiotemporal precision and minimal crosstalk using existing methods remains challenging. Here, we report a soft neural probe for multiplexed neurochemical monitoring via the electrografting-assisted site-selective functionalization of aptamers on graphene field-effect transistors (G-FETs). The neural probes possess excellent flexibility, ultralight mass (28 mg), and a nearly cellular-scale dimension of 50 µm × 50 µm for each G-FET. As a demonstration, we show that G-FETs with electrochemically grafted molecular linkers (-COOH or -NH2) and specific aptamers can be used to monitor serotonin and dopamine with high sensitivity (limit of detection: 10 pM) and selectivity (dopamine sensor >22-fold over norepinephrine; serotonin sensor >17-fold over dopamine). In addition, we demonstrate the feasibility of the simultaneous monitoring of dopamine and serotonin in a single neural probe with minimal crosstalk and interferences in phosphate-buffered saline, artificial cerebrospinal fluid, and harvested mouse brain tissues. The stability studies show that multiplexed neural probes maintain the capability for simultaneously monitoring dopamine and serotonin with minimal crosstalk after incubating in rat cerebrospinal fluid for 96 h, although a reduced sensor response at high concentrations is observed. Ex vivo studies in harvested mice brains suggest potential applications in monitoring the evoked release of dopamine and serotonin. The developed multiplexed detection methodology can also be adapted for monitoring other neurochemicals, such as metabolites and neuropeptides, by simply replacing the aptamers functionalized on the G-FETs.

Controlling the Biological Behaviors of Polymer-Coated Upconverting Nanoparticles by Adjusting the Linker Length of Estrone Ligands.

The estrone ligand is used for modifying nanoparticle surfaces to improve their targeting effect on cancer cell lines. However, to date, there is no common agreement on the ideal linker length to be used for the optimum targeting performance. In this study, we aimed to investigate the impact of poly(poly ethylene glycol methyl ether methacrylate) (PPEGMEMA) linker length on the cellular uptake behavior of polymer-coated upconverting nanoparticles (UCNPs). Different triblock terpolymers, poly(poly (ethylene glycol) methyl ether methacrylate)-block-polymethacrylic acid-block-polyethylene glycol methacrylate phosphate (PPEGMEMAx-b-PMAAy-b-PEGMP3: x = 7, 15, 33, and 80; y = 16, 20, 18, and 18), were synthesized with different polymer linker chain lengths between the surface and the targeting ligand by reversible addition-fragmentation chain transfer polymerization. The estrone ligand was attached to the polymer via specific terminal conjugation. The cellular association of polymer-coated UCNPs with linker chain lengths was evaluated in MCF-7 cells by flow cytometry. Our results showed that the bioactivity of ligand modification is dependent on the length of the polymer linker. The shortest polymer PPEGMEMA7-b-PMAA16-b-PEGMP3 with estrone at the end of the polymer chain was found to have the best cellular association behavior in the estrogen receptor (ER)α-positive expression cell line MCF-7. Additionally, the anticancer drug doxorubicin•HCl was encapsulated in the nanocarrier to evaluate the 2D and 3D cytotoxicity. The results showed that estrone modification could efficiently improve the cellular uptake in ERα-positive expression cell lines and in 3D spheroid models.

Evidence for surface effects on the intermolecular interactions in Fe(II) spin crossover coordination polymers.

From X-ray absorption spectroscopy (XAS) and X-ray photoemission spectroscopy (XPS), it is evident that the spin state transition behavior of Fe(II) spin crossover coordination polymer crystallites at the surface differs from the bulk. A comparison of four different coordination polymers reveals that the observed surface properties may differ from bulk for a variety of reasons. There are Fe(II) spin crossover coordination polymers with either almost complete switching of the spin state at the surface or no switching at all. Oxidation, differences in surface packing, and changes in coordination could all contribute to making the surface very different from the bulk. Some Fe(II) spin crossover coordination polymers may be sufficiently photoactive so that X-ray spectroscopies cannot discern the spin state transition.

Coordination Chemistry of Uranyl Ions with Surface-Immobilized Peptides: An XPS Study.

The coordination chemistry of uranyl ions with surface immobilized peptides was studied using X-ray photoemission spectroscopy (XPS). All the peptides in the study were modified using a six-carbon alkanethiol as a linker on a gold substrate with methylene blue as the redox label. The X-ray photoemission spectra reveal that each modified peptide interacts differently with the uranyl ion. For all the modified peptides, the XPS spectra were taken in both the absence and presence of the uranium, and their comparison reveals that the interaction depends on the chemical group present in the peptides. The XPS results show that, among all the modified peptides in the current study, the (arginine)9 (R9) modified peptide showed the largest response to uranium. In the order of response to uranium, the second largest response was shown by the modified (arginine)6 (R6) peptide followed by the modified (lysine)6 (K6) peptide. Other modified peptides, (alanine)6 (A6), (glutamic acid)6 (E6) and (serine)6 (S6), did not show any response to uranium.

Application of Calcium-Binding Motif of E-Cadherin for Electrochemical Detection of Pb(II).

We report, for the first time, the use of a 13-amino-acid peptide sequence derived from the calcium-binding site of E-cadherin in the fabrication of an electrochemical peptide-based (E-PB) Pb(II) sensor. The sensing mechanism is analogous to that of previously developed E-PB sensors. Binding of Pb(II) rigidifies the surface-immobilized and methylene blue (MB)-modified peptide probe, thereby limiting the accessibility of the tethered MB to the electrode surface. This change in probe flexibility results in a reduction in the MB current that is dependent on the target concentration. The sensor behaves as a "signal-off" sensor in alternating current voltammetry and cyclic voltammetry, but it can behave as a "signal-on" sensor in differential pulse voltammetry when a longer pulse width is employed. It is capable of specific detection of Pb(II) and is selective enough to be employed in realistically complex samples such as diluted tap water, saliva, and urine samples. The detection is fast; signal saturation can be achieved in <60 s. The sensor can also be fabricated on gold-plated screen-printed carbon electrodes, electrode substrates that are ideal for cost-effective analysis of Pb(II) in real-world settings.

Tunable Signal-Off and Signal-On Electrochemical Cisplatin Sensor.

We report the first electrochemical cisplatin sensor fabricated with a thiolated and methylene blue (MB)-modified oligo-adenine (A)-guanine (G) DNA probe. Depending on the probe coverage, the sensor can behave as a signal-off or signal-on sensor. For the high-coverage sensor, formation of intrastrand Pt(II)-AG adducts rigidifies the oligo-AG probe, resulting in a concentration-dependent decrease in the MB signal. For the low-coverage sensor, the increase in probe-to-probe spacing enables binding of cisplatin via the intrastrand GNG motif (N = A), generating a bend in the probe which results in an increase in the MB current. Although both high-coverage signal-off and low-coverage signal-on sensors are capable of detecting cisplatin, the signal-on sensing mechanism is better suited for real time analysis of cisplatin. The low-coverage sensor has a lower limit of detection, wider optimal AC frequency range, and faster response time. It has high specificity for cisplatin and potentially other Pt(II) drugs and does not cross-react with satraplatin, a Pt(IV) prodrug. It is also selective enough to be employed directly in 50% saliva and 50% urine. This detection strategy may offer a new approach for sensitive and real time analysis of cisplatin in clinical samples.

Hexavalent Chromium as an Electrocatalyst in DNA Sensing.

We report for the first time the use of hexavalent chromium (Cr(VI)) as an electrocatalyst in electrochemical DNA sensing. For both stem-loop probe and linear probe electrochemical DNA sensors, the increase in probe rigidity upon target hybridization alters the accessibility of Cr(VI) to the methylene blue label on the surface-immobilized DNA probes. This change results in an enhancement in the electrocatalytic current when the sensors are interrogated using cyclic voltammetry at a slow scan rate. The incorporation of this electrocatalyst does not affect the normal "signal-off" sensing behavior observed in alternating current voltammetry; instead it enables simultaneous "signal-on" and "signal-off" detection of the target, while maintaining noted attributes of this class of folding- and dynamics-based sensors such as reusability and high selectivity. It is also capable of improving the limit of detection of the sensors by an order of magnitude. Importantly, this accessibility-based electrocatalytic sensing strategy is versatile and can potentially be used with other folding- and dynamics-based electrochemical biosensors.

Electrochemical Gold(III) Sensor with High Sensitivity and Tunable Dynamic Range.

We report the design and fabrication of a sensitive, specific, and selective electrochemical ion (E-ION) sensor for detection of Au(III). The signaling mechanism is based on the interactions between Au(III) and adenine; formation of these complexes rigidifies the methylene blue (MB)-modified oligoadenine probes, resulting in a concentration-dependent reduction in the MB signal. The dynamic range of the sensor can be tuned by simply changing the length of the DNA probe (six (A6) or 12 (A12) adenines). Independent of the probe length, both sensors have demonstrated to be sensitive, with a limits of detection of 50 and 20 nM for the A6 and A12 sensors, respectively. With further optimization, this sensing strategy may offer a promising approach for analyzing Au(III).

Electrochemical Detection of Platinum(IV) Prodrug Satraplatin in Serum.

We report the design and fabrication of a reagentless and reusable electrochemical sensor for detection of satraplatin (SAT), a platinum(IV) prodrug. The detection strategy is based on the electrocatalytic reaction between the Pt(IV) center of SAT and surface-immobilized methylene blue. We systematically evaluated the effect of passivating diluent chain length on the overall sensor performance. Our results show that the use of a shorter diluent like 2-mercaptoethanol is more advantageous than using a longer and more passivating diluent such as 6-mercapto-1-hexanol. Independent of the use of cyclic voltammetry or chronoamperometry as the sensor interrogation technique, all three sensors, each passivated with a different alkanethiol diluent, have been demonstrated to be sensitive; the limit of detection is in the range of 1-10 µM. They are also highly specific and do not respond to Pt(II) drugs such as cisplatin and carboplatin. More importantly, they are selective enough to be employed directly in 50% serum. This sensing strategy has potential applications in clinical pharmacokinetics studies.

Methylene blue-mediated electrocatalytic detection of hexavalent chromium.

We report, for the first time, the design and fabrication of an electrochemical ion (E-ION) sensor for highly specific detection of hexavalent chromium (Cr(VI)). Unlike previously developed electrochemical Cr(VI) sensors, the sensing mechanism relies on the previously unexplored electrocatalytic reaction between Cr(VI) and surface-immobilized methylene blue (MB). The sensor is sensitive, specific, and selective enough to be used in a synthetic aquifer sample. Like many sensors of this class, it is also reagentless, reusable, and compatible with gold-plated screen-printed carbon electrodes. Despite the difference in the sensing mechanism, this E-ION Cr(VI) sensor possesses attributes similar to other MB-based electrochemical sensors, sensors with potential for real world applications.

Comparison of Mannose, Ethylene Glycol, and Methoxy-Terminated Diluents on Specificity and Selectivity of Electrochemical Peptide-Based Sensors.

We report the synthesis and application of three new antifouling diluents for the fabrication of an E-PB HIV sensor. Among the three thiolated antifouling diluents used in this study, the methoxy-terminated diluent (C6-MEG) is the most effective in alleviating both nonspecific binding and adsorption of matrix contaminants onto the sensor surface, especially when compared to the mannose- (C6-MAN) and ethylene-glycol-terminated (C6-EG) diluents. The sensor fabricated with C6-MEG has a specificity factor (∼13.5) substantially higher than the sensor passivated with only 6-mercapto-1-hexanol (∼1.5). It is functional even when employed directly in 25% serum, an achievement that has not been observed with this class of E-PB sensors. More importantly, incorporation of these antifouling diluents has negligible impact on other important sensor properties such as sensitivity and binding kinetics. This sensor passivation strategy is versatile and can potentially be used with other E-PB sensors, as well as surface-based sensors that utilize thiol-gold self-assembled monolayer chemistry.

Fabrication of electrochemical DNA sensors on gold-modified recessed platinum nanoelectrodes.

We report the use of gold-modified recessed platinum (Pt) nanoelectrodes in the fabrication of linear and stem-loop probe-based electrochemical DNA (E-DNA) sensors. Pt nanoelectrodes with a radius less than 10 nm were reproducibly fabricated using an optimized laser pulling technique. Prior to sensor fabrication, the nanoelectrode was electrochemically etched to create a recessed nanopore, followed by electrodeposition of gold into the nanopore using either cyclic voltammetry or constant potential amperometry. Both techniques enabled controlled deposition of gold into the nanopores, resulting in a nanostructured gold electrode with a well-defined surface area. In addition, we systematically determined the optimal experimental condition for DNA probe immobilization and target interrogation. The electron transfer rate constants of methylene blue, as determined using alternating current voltammetry, were found to be much higher than those obtained from E-DNA sensors fabricated on conventional macroscale electrodes. While this unique phenomenon requires further investigation, our results clearly show that these gold-modified nanoelectrodes can be used as substrates for this class of electrochemical biosensors.

Effects of DNA probe and target flexibility on the performance of a "signal-on" electrochemical DNA sensor.

We report the effect of the length and identity of a nontarget binding spacer in both the probe and target sequences on the overall performance of a folding-based electrochemical DNA sensor. Six near-identical DNA probes were used in this study; the main differences between these probes are the length (6, 10, or 14 bases) and identity (thymine (T) or adenine (A)) of the spacer connecting the two target binding domains. Despite the differences, the signaling mechanism of these sensors remains essentially the same. The methylene blue (MB)-modified probe assumes a linear unstructured conformation in the absence of the target; upon hybridization to the target, the probe adopts a "close" conformation, resulting in an increase in the MB current. Among the six sensors, the T14 and A14 sensors showed the largest signal increase upon target hybridization, highlighting the significance of probe flexibility on sensor performance. In addition to the target without a midsequence spacer, 12 other targets, each with a different oligo-T or oligo-A spacer, were used to elucidate the effect of target flexibility on the sensors' signaling capacity. For all six sensors, hybridization to targets with a 2- or 3-base spacer resulted in the largest signal increase. Higher signal enhancement was also observed with targets with an oligo-A spacer. For this sensor design, addition of a long nontarget binding spacer to the probe sequence is advantageous, as it provides flexibility for optimal target capture. The length of the spacer in the target sequence, however, should be adequately long to enable efficient hybridization yet does not introduce undesirable electrostatic and crowding effects.

Electrochemical techniques for characterization of stem-loop probe and linear probe-based DNA sensors.

Here we present a summary of the sensor performance of the stem-loop probe (SLP) and linear probe (LP) electrochemical DNA sensors when interrogated using alternating current voltammetry (ACV), cyclic voltammetry (CV), and differential pulse voltammetry (DPV). Specifically, we identified one critical parameter for each voltammetric technique that can be adjusted for optimal sensor performance. Overall, the SLP sensor displayed good sensor performance (i.e., 60+% signal attenuation in the presence of the target) over a wider range of experimental conditions when compared to the LP sensor. When used with ACV, the optimal frequency range was found to be between 5 and 5000 Hz, larger than the 5-100 Hz range observed with the LP sensor. A similar trend was observed for the two sensors in CV; the LP sensor was operational only at scan rates between 30 and 100 V/s, whereas the SLP sensor performed well at scan rates between 1 and 1000 V/s. Unlike ACV and CV, DPV has demonstrated to be a more versatile sensor interrogation technique for this class of sensors. Despite the minor differences in total signal attenuation upon hybridization to the target DNA, both SLP and LP sensors performed optimally under most pulse widths used in this study. More importantly, when used with longer pulse widths, both sensors showed "signal-on" behavior, which is generally more desirable for sensor applications.

Polymersomes with micellar patches.

Hollow block copolymer particles called polymer vesicles (polymersomes) serve as versatile containers for compartmentalization in synthetic biology and drug delivery. Recently, there has been growing interest in using polymersomes as colloidal building blocks for creating higher-order clustered structures. Most reports thus far rely on the use of DNA base-pairing interactions to "glue" polymersomes with other colloidal components. In this study, we present two alternative electrostatically driven approaches to assemble polymersomes and model colloids (micelles) into hybrid clusters. The first approach uses pH to manipulate electrostatic interactions and effectively control the clustering extent of micellar subunits on polymersomes, while the second approach relies on the hydrolysis of an acid trigger, glucono delta-lactone (GDL), to introduce temporal control over clustering. We envisage our approaches and structures reported herein will help inspire the creation of new prospects for materials science and biomedical applications.

An interpretation for the components of 2p3/2core level x-ray photoelectron spectra of the cations in some inverse spinel oxides.

In an effort to reconcile the various interpretations for the cation components of the 2p3/2observed in x-ray photoelectron spectroscopy (XPS) of several spinel oxide materials, the XPS spectra of both spinel alloy nanoparticles and crystalline thin films are compared. We observed that different components of the 2p3/2core level XPS spectra, of these inverse spinel thin films, are distinctly surface and bulk weighted, indicating surface-to-bulk core level shifts in the binding energies. Surface-to-bulk core level shifts in binding energies of Ni and Fe 2p3/2core levels of NiFe2O4thin film are observed in angle-resolved XPS. The ratio between surface-weighted components and bulk-weighted components of the Ni and Fe core levels shows appreciable dependency on photoemission angle, with respect to surface normal. XPS showed that the ferrite nanoparticles NixCo1-xFe2O4(x= 0.2, 0.5, 0.8, 1) resemble the surface of the NiFe2O4thin film. Surface-to-bulk core level shifts are also observed in CoFe2O4and NiCo2O4thin films but not as significantly as in NiFe2O4thin film. Estimates of surface stoichiometry of some spinel oxide nanoparticles and thin films suggested that the apportionment between cationic species present could be farther from expectations for thin films as compared to what is seen with nanoparticles.

Effect of signaling probe conformation on sensor performance of a displacement-based electrochemical DNA sensor.

Here we report the effect of the signaling probe conformation on sensor performance of a "signal-on" folding-based electrochemical DNA sensor. The sensor is comprised of a methylene blue (MB)-modified signaling probe and an unlabeled capture probe that partially hybridize to each other at the distal end. In presence of the full-complement target which binds to the unlabeled capture probe, the labeled signaling probe is released. Two different signaling probes were used in this study, in which one is capable of assuming a stem-loop conformation (SLP-MB), whereas the other probe adopts a flexible linear conformation (LP-MB). In the presence of the full complement target DNA, both sensors showed a large increase in MB current when interrogated using alternating current (ac) voltammetry, verifying the release of the signaling probe. Overall, the SLP-MB sensor showed higher % signal enhancement; the LP-MB sensor, however, showed distinctly faster binding kinetics when interrogated under the same experimental conditions. The SLP-MB sensor displayed a wider usable ac frequency range when compared to the LP-MB sensor. Despite these differences, the detection limit and dynamic range were found to be similar among the two sensors. In addition to 6-mercapto-1-hexanol, longer chain hydroxyl-terminated alkanethiols were used to construct these sensors. Our results showed that sensors fabricated with longer chain diluents, independent of the sensor architecture, were not only functional, the signaling capability was significantly enhanced.

Use of combined scanning electrochemical and fluorescence microscopy for detection of reactive oxygen species in prostate cancer cells.

Release of ROS from prostate cancer (PC3) cells was studied using scanning electrochemical microscopy (SECM) and fluorescence microscopy. One-directional lateral scan SECM was used as a rapid and reproducible tool for simultaneous mapping of cell topography and reactive oxygen species (ROS) release. Fluorescence microscopy was used in tandem to monitor the tip position, in addition to providing information on intracellular ROS content via the use of ROS-reactive fluorescent dyes. A unique tip current (iT) vs lateral distance profile was observed when the tip potential (ET) was set at -0.65 V. This profile reflects the combined effects of topographical change and ROS release at the PC3 cell surfaces. Differentiation between topographical-related and ROS-induced current change was achieved by comparing the scans collected at -0.65 and -0.85 V. The effects of other parameters such as tip to cell distance, solvent oxygen content, and scan direction on the profile of the scan were systematically evaluated. Cells treated with tert-butyl hydroperoxide, a known ROS stimulus, were also evaluated using the lateral scanning approach. Overall, the SECM results correlate well with the fluorescence results. The extracellular ROS level detected at the SECM tip was found to be similar to the intracellular ROS level monitored using fluorescence microscopy. While the concentration of each contributing ROS species has not been determined and is thus part of the future study, here we have successfully demonstrated the use of a simple two-potential lateral scan approach for analysis of ROS released by living cells under real physiological conditions.