The Detection of Trace Metal Contaminants in Organic Products Using Ion Current Rectifying Quartz Nanopipettes.

While ion current rectification (ICR) in aprotic solvent has been fundamentally studied, its application in sensing devices lacks exploration. The development of sensors operable in these solvents is highly beneficial to the chemical industry, where polar aprotic solvents, such as acetonitrile, are widely used. Currently, this industry relies on the use of inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectroscopy (OES) for the detection of metal contamination in organic products. Herein, we present the detection of trace amounts of Pd2+ and Co2+ using ion current rectification, in cyclam-functionalized quartz nanopipettes, with tetraethylammonium tetrafluoroborate (TEATFB) in MeCN as supporting electrolyte. This methodology is employed to determine the concentration of Pd in organic products, before and after purification by Celite filtration and column chromatography, obtaining comparable results to ICP-MS within minutes and without complex sample preparation. Finite element simulations are used to support our experimental findings, which reveal that the formation of double-junction diodes in the nanopore enables trace detection of these metals, with a significant response from baseline even at picomolar concentrations.

Reproducibility and stability of silane layers in nanoconfined electrochemical systems.

Organosilanes are commonly utilized to attach bioreceptors to oxide surfaces. The deposition of such silane layers is especially challenging in nanoscale or nanoconfined devices, such as in nanopipettes, since rinsing off loosely bound silanes may not be possible due to geometric constrictions and because the thickness of multilayered silanes can cover or block nanoscale features. Furthermore, in electrochemical devices, the silane layers experience additional perturbations, such as electric migration and electroosmotic force. Despite its importance, there appears to be no consensus in the current literature on the optimal methodology for nanopipette silanization, with significant variations in reported conditions. Herein, we systematically investigate the reproducibility and stability of liquid- and vapor-phase deposited silane layers inside nanopipettes. Electrochemical monitoring of the changing internal silanized surface reveals that vapor-deposited APTES generates surface modifications with the highest reproducibility, while vapor-deposited APTMS generates surface modifications of the highest stability over a 24-hour time period. Practical issues of silanizing nanoconfined systems are highlighted, and the importance of carefully chosen silanization conditions to yield stable and reproducible monolayers is emphasized as an underappreciated aspect in the development of novel nanoscale systems.

A Visible-Light-Regulated Chloride Transport Channel Inspired by Rhodopsin.

Inspired by the light-regulating capabilities of naturally occurring rhodopsin, we have constructed a visible-light-regulated Cl- -transport membrane channel based on a supramolecular host-guest interaction. A natural retinal chromophore, capable of a visible-light response, is used as the guest and grafted into the artificial channel. Upon introduction of an ethyl-urea-derived pillar[6]arene (Urea-P6) host, threading or de-threading of the retinal and selective bonding of Cl- can be utilized to regulate ion transport. Based on the visible-light responsiveness of the host-guest interaction, Cl- transport can be regulated by visible light between ON and OFF states. Visible-light-regulated Cl- transport as a chemical model permits to understand comparable biological ion-selective transport behaviors. Furthermore, this result also supplies a smart visible-light-responsive Cl- transporter, which may have applications in natural photoelectric conversion and photo-controlled delivery systems.

Construction of A High-Flux Protein Transport Channel Inspired by the Nuclear Pore Complex.

Inspired by the nuclear pore complex (NPC), herein we have established a biomimetic high-flux protein delivery system via the ingenious introduction of pillar[5]arene-based host-guest system into one side of artificial hour-glass shaped nanochannel. With a transport flux of 660 lysozymes per minute, the system provides efficient high-flux protein transport at a rate which is significantly higher than that of an unmodified nanochannel and conventional bilateral symmetrical modified nanochannels. In view of these promising results, the use of artificial nanochannel to improve protein transport not only presents a new potential chemical model for biological research and better understanding of protein transport behavior in the living systems, but also provides a high-flux protein transporter device, which may have applications in the design of protein drug release systems, protein separation systems and microfluidics in the near future.

Hyperbaric oxygen is still needed in the management and prevention of mandibular necrosis: a response to Mr. Richard Clarke.

Mr. Richard Clarke presents in this Journal his arguments against continued application of hyperbaric oxygen (HBO2) therapy to the pre-extraction neoadjuvant treatment or the treatment of frank mandibular ORN. In the same article he advocates a promising renewed interest in HBO2 as a radiosensitizer. Arguments against HBO2 prior to extractions are based on several papers which consistently include low-risk patients. The just-released HOPON trial reports a negative pre-extraction outcome for HBO2, but patients were enrolled with radiation doses as low as 50Gy. For advanced mandibular necrosis (Marx Stage III) requiring resection, fibular free flap reconstruction is advocated. A high complication rate with free flaps is acknowledged but the magnitude of these complications is not discussed. A cost savings for this procedure is suggested, but no mention is made of the typical cost of the procedure ($90,000) or the requirement of a typical one-week hospital stay, including an initial one or two days in the ICU. Nor is mention made of the very low rate of subsequent dental rehabilitation. The success reported by Delainian, et al. employing pentoxifylline, Vitamin E and sometimes a bisphosphonate is equated to the four decades of HBO2 success with the Marx protocol for Stage I and II ORN. In the phase II trial by Delainian (not randomized) six of her 54 patients died secondary to sepsis, and she graded patients as complete responders if 5mm or less bone was exposed. Even at entry patients had an average of only 1.7 cm exposed bone and treatment was prolonged (16 + or -9 months). Any cost comparison studies will have to account for the indirect expenses of this prolonged treatment including lost productivity.

Review of medical radiography and tomography with proton beams.

The use of hadron beams, especially proton beams, in cancer radiotherapy has expanded rapidly in the past two decades. To fully realize the advantages of hadron therapy over traditional x-ray and gamma-ray therapy requires accurate positioning of the Bragg peak throughout the tumor being treated. A half century ago, suggestions had already been made to use protons themselves to develop images of tumors and surrounding tissue, to be used for treatment planning. The recent global expansion of hadron therapy, coupled with modern advances in computation and particle detection, has led several collaborations around the world to develop prototype detector systems and associated reconstruction codes for proton computed tomography (pCT), as well as more simple proton radiography, with the ultimate intent to use such systems in clinical treatment planning and verification. Recent imaging results of phantoms in hospital proton beams are encouraging, but many technical and programmatic challenges remain to be overcome before pCT scanners will be introduced into clinics. This review introduces hadron therapy and the perceived advantages of pCT and proton radiography for treatment planning, reviews its historical development, and discusses the physics related to proton imaging, the associated experimental and computation issues, the technologies used to attack the problem, contemporary efforts in detector and computational development, and the current status and outlook.

Dynamics of a DNA Mismatch Site Held in Confinement Discriminate Epigenetic Modifications of Cytosine.

The identification and discrimination of four epigenetic modifications to cytosine in the proposed active demethylation cycle is demonstrated at the single-molecule level, without the need for chemical pretreatment or labeling. The wild-type protein nanopore α-hemolysin is used to capture individual DNA duplexes containing a single cytosine-cytosine mismatch. The mismatch is held at the latch constriction of α-hemolysin, which is used to monitor the kinetics of base-flipping at the mismatch site. Base-flipping and the subsequent interactions between the DNA and the protein are dramatically altered when one of the cytosine bases is replaced with methyl-, hydroxymethyl-, formyl-, or carboxylcytosine. As well as providing a route to single-molecule analysis of important epigenetic markers in DNA, our results provide important insights into how the introduction of biologically relevant, but poorly understood, modifications to cytosine affect the local conformational dynamics of a DNA duplex in a confined environment.

Base Flipping within the α-Hemolysin Latch Allows Single-Molecule Identification of Mismatches in DNA.

A method for identifying and differentiating DNA duplexes containing the mismatched base pairs CC and CA at single molecule resolution with the protein pore α-hemolysin (αHL) is presented. Unique modulating current signatures are observed for duplexes containing the CC and CA mismatches when the mismatch site in the duplex is situated in proximity to the latch constriction of αHL during DNA residence inside the pore. The frequency and current amplitude of the modulation states are dependent on the mismatch type (CC or CA) permitting easy discrimination of these mismatches from one another, and from a fully complementary duplex that exhibits no modulation. We attribute the modulating current signatures to base flipping and subsequent interaction with positively charged lysine residues at the latch constriction of αHL. Our hypothesis is supported by the extended residence times of DNA duplexes within the pore when a mismatch is in proximity to the latch constriction, and by the loss of the two-state current signature in low pH buffers (<6.3), where the protonation of one of the cytosine bases increases the stability of the intrahelical state.

Energetics of base flipping at a DNA mismatch site confined at the latch constriction of α-hemolysin.

Unique, two-state modulating current signatures are observed when a cytosine-cytosine mismatch pair is confined at the 2.4 nm latch constriction of the α-hemolysin (αHL) nanopore. We have previously speculated that the modulation is due to base flipping at the mismatch site. Base flipping is a biologically significant mechanism in which a single base is rotated out of the DNA helical stack by 180°. It is the mechanism by which enzymes are able to access bases for repair operations without disturbing the global structure of the helix. Here, temperature dependent ion channel recordings of individual double-stranded DNA duplexes inside αHL are used to derive thermodynamic (ΔH, ΔS) and kinetic (EA) parameters for base flipping of a cytosine at an unstable cytosine-cytosine mismatch site. The measured activation energy for flipping a cytosine located at the latch of αHL out of the helix (18 ± 1 kcal mol-1) is comparable to that previously reported for base flipping at mismatch sites from NMR measurements and potential mean force calculations. We propose that the αHL nanopore is a useful tool for measuring conformational changes in dsDNA at the single molecule level.

A Fast Experimental Scanner for Proton CT: Technical Performance and First Experience with Phantom Scans.

We report on the design, fabrication, and first tests of a tomographic scanner developed for proton computed tomography (pCT) of head-sized objects. After extensive preclinical testing, pCT is intended to be employed in support of proton therapy treatment planning and pre-treatment verification in patients undergoing particle-beam therapy. The scanner consists of two silicon-strip telescopes that track individual protons before and after the phantom, and a novel multistage scintillation detector that measures a combination of the residual energy and range of the proton, from which we derive the water equivalent path length (WEPL) of the protons in the scanned object. The set of WEPL values and the associated paths of protons passing through the object over a 360° angular scan are processed by an iterative, parallelizable reconstruction algorithm that runs on modern GP-GPU hardware. In order to assess the performance of the scanner, we have performed tests with 200 MeV protons from the synchrotron of the Loma Linda University Medical Center and the IBA cyclotron of the Northwestern Medicine Chicago Proton Center. Our first objective was calibration of the instrument, including tracker channel maps and alignment as well as the WEPL calibration. Then we performed the first CT scans on a series of phantoms. The very high sustained rate of data acquisition, exceeding one million protons per second, allowed a full 360° scan to be completed in less than 10 minutes, and reconstruction of a CATPHAN 404 phantom verified accurate reconstruction of the proton relative stopping power in a variety of materials.

Detection of benzo[a]pyrene-guanine adducts in single-stranded DNA using the α-hemolysin nanopore.

The carcinogenic precursor benzo[a]pyrene (BP), a polycyclic aromatic hydrocarbon, is released into the environment through the incomplete combustion of hydrocarbons. Metabolism of BP in the human body yields a potent alkylating agent (benzo[a]pyrene diol epoxide, BPDE) that reacts with guanine (G) in DNA to form an adduct implicated in cancer initiation. We report that the α-hemolysin (αHL) nanopore platform can be used to detect a BPDE adduct to G in synthetic oligodeoxynucleotides. Translocation of a 41-mer poly-2'-deoxycytidine strand with a centrally located BPDE adduct to G through αHL in 1 M KCl produces a unique multi-level current signature allowing the adduct to be detected. This readily distinguishable current modulation was observed when the BPDE-adducted DNA strand translocated from either the 5' or 3' directions. This study suggests that BPDE adducts and other large aromatic biomarkers can be detected with αHL, presenting opportunities for the monitoring, quantification, and sequencing of mutagenic compounds from cellular DNA samples.

Temperature and electrolyte optimization of the α-hemolysin latch sensing zone for detection of base modification in double-stranded DNA.

The latch region of the wild-type protein pore α-hemolysin (α-HL) constitutes a sensing zone for individual abasic sites (and furan analogs) in double-stranded DNA (dsDNA). The presence of an abasic site or furan within a DNA duplex, electrophoretically captured in the α-HL vestibule and positioned at the latch region, can be detected based on the current blockage prior to duplex unzipping. We investigated variations in blockage current as a function of temperature (12-35°C) and KCl concentration (0.15-1.0 M) to understand the origin of the current signature and to optimize conditions for identifying the base modification. In 1 M KCl solution, substitution of a furan for a cytosine base in the latch region results in an ∼ 8 kJ mol(-1) decrease in the activation energy for ion transport through the protein pore. This corresponds to a readily measured ∼ 2 pA increase in current at room temperature. Optimal resolution for detecting the presence of a furan in the latch region is achieved at lower KCl concentrations, where the noise in the measured blockage current is significantly lower. The noise associated with the blockage current also depends on the stability of the duplex (as measured from the melting temperature), where a greater noise in the measured blockage current is observed for less stable duplexes.

Meeting the detector challenges for pre-clinical proton and ion computed tomography.

Six decades after its conception, proton computed tomography (pCT) and proton radiography have yet to be used in medical clinics. However, good progress has been made on relevant detector technologies in the past two decades, and a few prototype pCT systems now exist that approach the performance needed for a clinical device. The tracking and energy-measurement technologies in common use are described, as are the few pCT scanners that are in routine operation at this time. Most of these devices still look like detector R&D efforts as opposed to medical devices, are difficult to use, are at least a factor of five slower than desired for clinical use, and are too small to image many parts of the human body. Recommendations are made for what to consider when engineering a pre-clinical pCT scanner that is designed to meet clinical needs in terms of performance, cost, and ease of use.

Base-excision repair activity of uracil-DNA glycosylase monitored using the latch zone of α-hemolysin.

Nanopores have been investigated as a simple and label-free tool to characterize DNA nucleotides when a ssDNA strand translocates through the constriction of the pore. Here, a wild-type α-hemolysin protein nanopore was used to monitor DNA repair enzyme activity based on base-specific interactions of dsDNA with the vestibule constriction "latch", a previously unrecognized sensing zone in α-hemolysin specific for dsDNA structure. The presence of a single abasic site within dsDNA that is in proximity to the latch zone (±2 nucleotides) results in a large increase in ion channel current, allowing accurate quantitation of the kinetics of base repair reactions involving an abasic site product. Taking advantage of the high resolution for abasic site recognition, the rate of uracil-DNA glycosylase hydrolysis of the N-glycosidic bond, converting 2'-deoxyuridine in DNA to an abasic site, was continuously monitored by electrophoretically capturing reaction substrate or product dsDNA in the ion channel vestibule. Our work suggests use of the nanopore as an enzymology tool and provides a means to identify single base structural changes in dsDNA.

Tracker Readout ASIC for Proton Computed Tomography Data Acquisition.

A unique CMOS chip has been designed to serve as the front-end of the tracking detector data acquisition system of a pre-clinical prototype scanner for proton computed tomography (pCT). The scanner is to be capable of measuring one to two million proton tracks per second, so the chip must be able to digitize the data and send it out rapidly while keeping the front-end amplifiers active at all times. One chip handles 64 consecutive channels, including logic for control, calibration, triggering, buffering, and zero suppression. It outputs a formatted cluster list for each trigger, and a set of field programmable gate arrays merges those lists from many chips to build the events to be sent to the data acquisition computer. The chip design has been fabricated, and subsequent tests have demonstrated that it meets all of its performance requirements, including excellent low-noise performance.

A label-free, electrochemical SERS-based assay for detection of DNA hybridization and discrimination of mutations.

A label-free, surface-enhanced Raman spectroscopy-based assay for detecting DNA hybridization at an electrode surface and for distinguishing between mutations in DNA is demonstrated. Surface-immobilized DNA is exposed to a binding agent that is selective for dsDNA and acts as a reporter molecule. Upon application of a negative potential, the dsDNA denatures into its constituent strands, and the changes in the spectra of the reporter molecule are monitored. This method has been used to distinguish between a wild-type, 1653C/T single-point mutation and ΔF508 triplet deletion in the CFTR gene. The use of dsDNA-selective binding agents as reporter molecules in a discrimination assay removes the burden of synthetically modifying the target to be detected, while retaining flexibility in the choice of the reporter molecule.

Real-time surface-enhanced Raman spectroscopy monitoring of surface pH during electrochemical melting of double-stranded DNA.

The application of a negative potential ramp at a double-stranded DNA (dsDNA) functionalized electrode surface results in the gradual denaturation of the DNA in a process known as electrochemical melting. The underlying physical chemistry behind electrochemically driven DNA denaturation is not well understood, and one possible mechanism is a change in local pH at the electrode surface. We demonstrate that by coimmobilization of p-mercaptobenozic acid at a dsDNA-functionalized electrode surface, it is possible to monitor both DNA denaturation and the local pH simultaneously using surface-enhanced Raman spectroscopy. We find that the local pH at the electrode surface does not change as the applied potential is scanned negative and the dsDNA denatures. We therefore conclude that in these experiments electrochemical melting is not caused by electrochemically driven local pH changes.

Aprotic Solvent Accumulation Amplifies Ion Current Rectification in Conical Nanopores.

Ion current rectification is highly reported in aqueous electrochemical systems and sensors but lacks exploration in organic systems due to the additional complexity introduced by non-aqueous solvents. Herein, a detailed study on ion current rectification with highly polar and mildly polar aprotic organic solvents as a function of tetraethylammonium tetrafluoroborate supporting electrolyte concentration is presented. To explain our experimental results, we introduce a previously unreported phenomenon: the formation of a double-junction diode within the nanopore that arises due to a complex interplay between ion and solvent enrichment effects. Finite element simulations are used to explore this phenomenon and the subsequent effect on the rectifying behavior of conical quartz nanopores.

Effect of Electrolyte Concentration and Pore Size on Ion Current Rectification Inversion.

A thorough understanding of nanoscale transport properties is vital for the development and optimization of nanopore sensors. The thickness of the electrical double layers (EDLs) at the internal walls of a nanopore, as well as the dimensions of the nanopore itself, plays a crucial role in determining transport properties. Herein, we demonstrate the effect of the electrolyte concentration, which is inversely proportional to the EDL thickness, and the effect of pore size, which controls the extent of the electrical double layer overlap, on the ion current rectification phenomenon observed for conical nanopores. Experimental and numerical results showed that as the electrolyte concentration is decreased, the rectification ratio reaches a maximum, then decreases, and eventually inverts below unity. We also show that as the pore size is decreased, the rectification maximum and the inversion take place at higher electrolyte concentrations. Numerical investigations revealed that both phenomena occur due to the shifting of ion enrichment distributions within the nanopore as the electrolyte concentration or the pore size is varied.

The role of Monte Carlo simulation in understanding the performance of proton computed tomography.

Proton computed tomography (pCT) is a promising tomographic imaging modality allowing direct reconstruction of proton relative stopping power (RSP) required for proton therapy dose calculation. In this review article, we aim at highlighting the role of Monte Carlo (MC) simulation in pCT studies. After describing the requirements for performing proton computed tomography and the various pCT scanners actively used in recent research projects, we present an overview of available MC simulation platforms. The use of MC simulations in the scope of investigations of image reconstruction, and for the evaluation of optimal RSP accuracy, precision and spatial resolution omitting detector effects is then described. In the final sections of the review article, we present specific applications of realistic MC simulations of an existing pCT scanner prototype, which we describe in detail.