Designing Molecularly Imprinted Polymer-Modified Boron-Doped Diamond Electrodes for Highly Selective Electrochemical Drug Sensors.

Drug detection in biological solutions is essential in studying the pharmacokinetics of the body. Electrochemical detection is an accurate and rapid method, but measuring multiple drugs that react at similar potentials is challenging. Herein, we developed an electrochemical sensor using a boron-doped diamond (BDD) electrode modified with a molecularly imprinted polymer (MIP) to provide specificity in drug sensing. The MIP is a polymer material designed to recognize and capture template molecules, enabling the selective detection of target molecules. In this study, we selected the anticancer drug doxorubicin (DOX) as the template molecule. In the electrochemical measurements using an unmodified BDD, the DOX reduction was observed at approximately -0.5 V (vs Ag/AgCl). Other drugs, i.e., mitomycin C or clonazepam (CZP), also underwent a reduction reaction at a similar potential to that of DOX, when using the unmodified BDD, which rendered the accurate quantification of DOX in a mixture challenging. Similar measurements conducted in PBS using the MIP-BDD only resulted in a DOX reduction current, with no reduction reaction observed in the presence of mitomycin C and CZP. These results suggest that the MIP, whose template molecule is DOX, inhibits the reduction of other drugs on the electrode surface. Selective DOX measurement using the MIP-BDD was also possible in human plasma, and the respective limits of detection of DOX in PBS and human plasma were 32.10 and 16.61 nM. The MIP-BDD was durable for use in six repeated measurements, and MIP-BDD may be applicable as an electrochemical sensor for application in therapeutic drug monitoring.

A strategy for low-cost portable monitoring of plasma drug concentrations using a sustainable boron-doped-diamond chip.

On-site monitoring of plasma drug concentrations is required for effective therapies. Recently developed handy biosensors are not yet popular owing to insufficient evaluation of accuracy on clinical samples and the necessity of complicated costly fabrication processes. Here, we approached these bottlenecks via a strategy involving engineeringly unmodified boron-doped diamond (BDD), a sustainable electrochemical material. A sensing system based on a ∼1 cm2 BDD chip, when analysing rat plasma spiked with a molecular-targeting anticancer drug, pazopanib, detected clinically relevant concentrations. The response was stable in 60 sequential measurements on the same chip. In a clinical study, data obtained with a BDD chip were consistent with liquid chromatography-mass spectrometry results. Finally, the portable system with a palm-sized sensor containing the chip analysed ∼40 µL of whole blood from dosed rats within ∼10 min. This approach with the 'reusable' sensor may improve point-of-monitoring systems and personalised medicine while reducing medical costs.

A rapid and simple electrochemical detection of the free drug concentration in human serum using boron-doped diamond electrodes.

Monitoring drug concentration in blood and reflecting this in the dosage are crucial for safe and effective drug treatment. Most drug assays are based on total concentrations of bound and unbound proteins in the serum, although only the unbound concentration causes beneficial and adverse events. Monitoring the unbound concentration alone is expected to provide a means for further optimisation of drug treatment. However, unbound concentration monitoring has not been routinely used for drug treatment due to the long analysis time and the high cost of conventional methods. Here, we have developed a rapid electrochemical method to determine the unbound concentration in ultrafiltered human serum using boron-doped diamond (BDD) electrodes. When the anticancer drug doxorubicin was used as the test drug, the catalytic doxorubicin-mediated reduction of dissolved oxygen provided a sensitive electrochemical signal, with a detection limit of 0.14 nM. In contrast, the sensitivity of glassy carbon (GC) was inferior under the same conditions due to interference from the dissolved oxygen reduction current. The signal background ratio (S/B) of BDD and GC was 11.5 (10 nM doxorubicin) and 1.1 (50 nM), respectively. The results show that a fast measurement time within ten seconds is possible in the clinical concentration range. Additionally, in the ultrafiltered human serum, the obtained values of unbound doxorubicin concentration showed good agreement with those quantified by conventional liquid chromatography-mass spectrometry. This approach has the potential for application in clinical settings where rapid and simple analysis methods would be beneficial.

Core-shell copper-gold nanoparticles modified at the boron-doped diamond electrode for oxygen sensors.

Bimetallic copper-gold (Cu@Au) nanoparticles were synthesized and utilised to modify boron-doped diamond (BDD) electrodes. Nanorod particles with a diameter size of around 10 nm and a length of around 20 nm were successfully synthesized. These nanoparticles were then attached to the BDD surface by using allylamine as the bridge. Comparison among the BDD modified with Cu@Au and individual gold nanoparticles showed that Cu@Au nanoparticles created around 3 times higher gold coverage on the BDD surface than normal gold nanoparticles. It was also found that the use of allylamine as the bridge can attach more gold than copper nanoparticles. Moreover, around two times higher current responses of oxygen reduction reaction were observed at Cu@Au-modified BDD. Good linearity in a concentration range from 2 to 9 ppm could be achieved with a sensitivity of 0.0138 mA ppm-1 and limit detection of 1.98 ppm. An application of the modified BDD for a biochemical oxygen demand (BOD) sensor using Rhodotorula mucilaginosa UICC Y-181 as the biosensing agent was also demonstrated with glucose solutions as the solution model. Sensitivity equivalent to 17.4 µA mM-1 BOD could be achieved. The system showed good stability with an RSD of 3.45% in 10 measurements.

Nanodiamonds Inhibit Cancer Cell Migration by Strengthening Cell Adhesion: Implications for Cancer Treatment.

Nanodiamonds (NDs) are a type of biocompatible nanomaterial with easily modified surfaces and are considered as promising candidates in biomedicine. In this work, the inhibition of tumor cell migration by carboxylated nanodiamonds (cNDs) was investigated. AFM-based single cell adhesion and F-actin staining experiments demonstrated that cNDs treatment could enhance cell adhesion and impair assembly of the cytoskeleton. The mechanism analysis of the regulatory protein expression level also proved that cNDs could inhibit the migration of Hela cells by preventing the epithelial-mesenchymal transition (EMT) process through the transforming growth factor ß (TGF-ß) signaling pathway. The in vivo pulmonary metastasis model also showed that cNDs effectively reduced the metastasis of murine B16 melanoma cells. In summary, cNDs have been demonstrated to inhibit cancer cell migration in vitro and decrease tumor metastasis in vivo. Therefore, cNDs might have potential utility for specific cancer treatment.

Avian Influenza Virus Detection by Optimized Peptide Termination on a Boron-Doped Diamond Electrode.

The development of a simple detection method with high sensitivity is essential for the diagnosis and surveillance of infectious diseases. Previously, we constructed a sensitive biosensor for the detection of pathological human influenza viruses using a boron-doped diamond electrode terminated with a sialyloligosaccharide receptor-mimic peptide that could bind to hemagglutinins involved in viral infection. Circulation of influenza induced by the avian virus in humans has become a major public health concern, and methods for the detection of avian viruses are urgently needed. Here, peptide density and dendrimer generation terminated on the electrode altered the efficiency of viral binding to the electrode surface, thus significantly enhancing charge-transfer resistance measured by electrochemical impedance spectroscopy. The peptide-terminated electrodes exhibited an excellent detection limit of less than one plaque-forming unit of seasonal H1N1 and H3N2 viruses. Furthermore, the improved electrode was detectable for avian viruses isolated from H5N3, H7N1, and H9N2, showing the potential for the detection of all subtypes of influenza A virus, including new subtypes. The peptide-based electrochemical architecture provided a promising approach to biosensors for ultrasensitive detection of pathogenic microorganisms.

[Development of in vivo drug sensing system with needle-type diamond microelectrode].

Continuous and real-time measurement of local concentrations of systemically administered drugs in vivo must be crucial for pharmacological studies. Nevertheless, conventional methods require considerable samples quantity and have poor sampling rates. Additionally, they cannot determine how drug kinetics correlates with target function over time. Here, we describe a system with two different sensors. One is a needle-type microsensor composed of boron-doped diamond with a tip of ~40 µm in diameter, and the other is a glass microelectrode. We first tested bumetanide. This diuretic can induce deafness. In the guinea-pig cochlea injected intravenously with bumetanide, the changes of the drug concentration and the extracellular potential underlying hearing were simultaneously measured in real time. We further examined an antiepileptic drug lamotrigine in the rat brain, and tracked its kinetics and at the same time the local field potentials representing neuronal activity. The action of the anticancer reagent doxorubicin was also monitored in the cochlea. This microsensing system may be applied to analyze pharmacokinetics and pharmacodynamics of various drugs at local sites in vivo, and contribute to promoting the pharmacological researches.

Comparison of performance between boron-doped diamond and copper electrodes for selective nitrogen gas formation by the electrochemical reduction of nitrate.

The electrochemical nitrate reduction by using boron-doped diamond (BDD) and copper (Cu) electrodes was investigated at various potentials. Product selectivity of nitrate reduction was strongly dependent on the applied potential for both electrodes. The highest selectivity of nitrogen gas production was obtained at -2.0 V (vs. Ag/AgCl) by using a BDD electrode with a faradaic efficiency as high as 45.2%. Compared with Cu electrode, nitrate reduction on BDD electrode occurred at more positive potential, and the production of nitrogen gas was larger. The transformation of surface-adsorbed nitrate into molecular nitrogen would be accelerated on BDD electrode with hindering nitrite production. In addition, low concentration of surface-adsorbed hydrogen on the BDD would also retard the ammonia generation, leading to increase in the selectivity of nitrogen gas formation. Meanwhile, BDD electrode could hinder the hydrogen evolution reaction, which enhanced the efficiency for nitrate reduction and decreased energy consumption. BDD electrode has excellent stability to remain better performance for reducing nitrate during electrolysis without any variation of surface morphology or chemical components.

Phase 1 study of sulfasalazine and cisplatin for patients with CD44v-positive gastric cancer refractory to cisplatin (EPOC1407).

A previous dose-escalation study of sulfasalazine (SSZ), an inhibitor of cystine-glutamate exchange transporter xc (-), in the variant form of CD44 (CD44v)-positive cancer stem cells (CSCs) suggested that administration of SSZ induces the reduction of CD44v-positive cells and intracellular reduced glutathione (GSH) levels in patients with advanced gastric cancer (AGC). Here we report a study to evaluate SSZ in combination with cisplatin in patients with CD44v-expressing AGC refractory to cisplatin. SSZ was given by oral administration four times daily with 2 weeks on and 1 week off. Cisplatin at 60 mg/m2 was administered every 3 weeks. Of the 15 patients who underwent prescreening of CD44v expression, 8 patients were positive, and 7 patients were treated with the dose level of SSZ at 6 g/day. One patient experienced dose-limiting toxicity (DLT) as grade 3 anorexia. Although no other patients experienced DLT, 4 patients required dose interruption or reduction of SSZ; thus, we terminated further dose escalation. No patient achieved objective response, but 1 patient completed six cycles with stable disease for more than 4 months as well as reduction of intratumoral GSH level. The combination of SSZ plus cisplatin was manageable, although dose modification was frequently required during a short observational period.

Dose-escalation study for the targeting of CD44v+ cancer stem cells by sulfasalazine in patients with advanced gastric cancer (EPOC1205).

BACKGROUND: Cancer stem cells (CSCs) have enhanced mechanisms of protection from oxidative stress. A variant form of CD44 (CD44v), a major CSC marker, was shown to interact with xCT, a subunit of cystine-glutamate transporter, which maintains high levels of intracellular reduced glutathione (GSH) which defend the cell against oxidative stress. Sulfasalazine (SSZ) is an inhibitor of xCT and was shown to suppress the survival of CD44v-positive stem-like cancer cells both in vitro and in vivo. To find the dose of SSZ which can safely reduce the population of CD44v-positive cells in tumors, a dose-escalation study in patients with advanced gastric cancer was conducted. METHODS: SSZ was given four times daily by oral administration with 2 weeks as one cycle. Tumor biopsies were obtained before and after 14 days of administration of SSZ to evaluate expression of CD44v and the intratumoral level of GSH. RESULTS: Eleven patients were enrolled and received a dosage from 8 to 12 g/day. Safety was confirmed up to a dosage of 12 g/day, which was considered the maximum tolerated dose. Among the eight patients with CD44v-positive cells in their pretreatment biopsy samples, the CD44v-positive cancer cell population appeared to be reduced in the posttreatment biopsy tissues of four patients. Intratumoral GSH levels were also decreased in two patients, suggesting biological effectiveness of SSZ at 8 g/day or greater. CONCLUSIONS: This is the first study of SSZ as an xCT inhibitor for targeting CSCs. Reduction of the levels of CD44v-positive cells and GSH was observed in some patients, consistent with the mode of action of SSZ in CSCs.

A microsensing system for the in vivo real-time detection of local drug kinetics.

Real-time recording of the kinetics of systemically administered drugs in in vivo microenvironments may accelerate the development of effective medical therapies. However, conventional methods require considerable analyte quantities, have low sampling rates and do not address how drug kinetics correlate with target function over time. Here, we describe the development and application of a drug-sensing system consisting of a glass microelectrode and a microsensor composed of boron-doped diamond with a tip of around 40 µm in diameter. We show that, in the guinea pig cochlea, the system can measure-simultaneously and in real time-changes in the concentration of bumetanide (a diuretic that is ototoxic but applicable to epilepsy treatment) and the endocochlear potential underlying hearing. In the rat brain, we tracked the kinetics of the drug and the local field potentials representing neuronal activity. We also show that the actions of the antiepileptic drug lamotrigine and the anticancer reagent doxorubicin can be monitored in vivo. Our microsensing system offers the potential to detect pharmacological and physiological responses that might otherwise remain undetected.

Highly sensitive detection of influenza virus by boron-doped diamond electrode terminated with sialic acid-mimic peptide.

The progression of influenza varies according to age and the presence of an underlying disease; appropriate treatment is therefore required to prevent severe disease. Anti-influenza therapy, such as with neuraminidase inhibitors, is effective, but diagnosis at an early phase of infection before viral propagation is critical. Here, we show that several dozen plaque-forming units (pfu) of influenza virus (IFV) can be detected using a boron-doped diamond (BDD) electrode terminated with a sialic acid-mimic peptide. The peptide was used instead of the sialyloligosaccharide receptor, which is the common receptor of influenza A and B viruses required during the early phase of infection, to capture IFV particles. The peptide, which was previously identified by phage-display technology, was immobilized by click chemistry on the BDD electrode, which has excellent electrochemical characteristics such as low background current and weak adsorption of biomolecules. Electrochemical impedance spectroscopy revealed that H1N1 and H3N2 IFVs were detectable in the range of 20-500 pfu by using the peptide-terminated BDD electrode. Our results demonstrate that the BDD device integrated with the receptor-mimic peptide has high sensitivity for detection of a low number of virus particles in the early phase of infection.

Yeast-based Biochemical Oxygen Demand Sensors Using Gold-modified Boron-doped Diamond Electrodes.

A gold nanoparticle modified boron-doped diamond electrode was developed as a transducer for biochemical oxygen demand (BOD) measurements. Rhodotorula mucilaginosa UICC Y-181 was immobilized in a sodium alginate matrix, and used as a biosensing agent. Cyclic voltammetry was applied to study the oxygen reduction reaction at the electrode, while amperometry was employed to detect oxygen, which was not consumed by the microorganisms. The optimum waiting time of 25 min was observed using 1-mm thickness of yeast film. A comparison against the system with free yeast cells shows less sensitivity of the current responses with a linear dynamic range (R(2) = 0.99) of from 0.10 mM to 0.90 mM glucose (equivalent to 10 - 90 mg/L BOD) with an estimated limit of detection of 1.90 mg/L BOD. However, a better stability of the current responses could be achieved with an RSD of 3.35%. Moreover, less influence from the presence of copper ions was observed. The results indicate that the yeast-immobilized BOD sensors is more suitable to be applied in a real condition.

High-yield electrochemical production of formaldehyde from CO2 and seawater.

The catalytic, electrocatalytic, or photocatalytic conversion of CO2 into useful chemicals in high yield for industrial applications has so far proven difficult. Herein, we present our work on the electrochemical reduction of CO2 in seawater using a boron-doped diamond (BDD) electrode under ambient conditions to produce formaldehyde. This method overcomes the usual limitation of the low yield of higher-order products, and also reduces the generation of H2 . In comparison with other electrode materials, BDD electrodes have a wide potential window and high electrochemical stability, and, moreover, exhibit very high Faradaic efficiency (74%) for the production of formaldehyde, using either methanol, aqueous NaCl, or seawater as the electrolyte. The high Faradaic efficiency is attributed to the sp(3)-bonded carbon of the BDD. Our results have wide ranging implications for the efficient and cost-effective conversion of CO2.

Controlling the diffusion profile of electroactive species for selective anodic stripping voltammetry of cadmium at boron-doped diamond electrodes.

Selective anodic stripping voltammetry of trace metal ions in a mixture solution with another interfering metal was developed based on Fick's law concerning the diffusion profile of interfering metals at the electrode surface after electrolysis treatment. A boron-doped diamond film was used as the sensing electrode, while a perforated carbon sheet was used for the interference-depleting electrode. The influence of the electrode distance and the time of electrolysis on the formation of the diffusion profile was studied. As a working model, the detection of cadmium with copper interference was investigated. The advantage of the method in comparison to general electrolysis was also discussed. The method offers a new perspective for improving the selective detection of metal ions by analyzing the diffusion profiles of the interfering species at the surface of electrodes.

In vivo assessment of cancerous tumors using boron doped diamond microelectrode.

The in vitro and in vivo electrochemical detection of the reduced form of glutathione (L-γ-glutamyl-L-cysteinyl-glycine, GSH) using boron doped diamond (BDD) microelectrode for potential application in the assessment of cancerous tumors is presented. Accurate calibration curve for the determination of GSH could be obtained by the in vitro electrochemical measurements. Additionally, it was shown that it was possible to separate the detection of GSH from the oxidized form of glutathione (GSSG) using chronoamperometry measurements. In vivo GSH detection measurements have been performed in human cancer cells inoculated in immunodeficient mice. These measurements have shown that the difference of GSH level between cancerous and normal tissues can be detected. Moreover, GSH detection measurements carried out before and after X-ray irradiation have proved that it is possible to assess in vivo the decrease in GSH concentration in the tumor after a specific treatment.

Development of a biochemical oxygen demand sensor using gold-modified boron doped diamond electrodes.

Gold-modified boron doped diamond (BDD) electrodes were examined for the amperometric detection of oxygen as well as a detector for measuring biochemical oxygen demand (BOD) using Rhodotorula mucilaginosa UICC Y-181. An optimum potential of -0.5 V (vs Ag/AgCl) was applied, and the optimum waiting time was observed to be 20 min. A linear calibration curve for oxygen reduction was achieved with a sensitivity of 1.4 µA mg(-1) L oxygen. Furthermore, a linear calibration curve in the glucose concentration range of 0.1-0.5 mM (equivalent to 10-50 mg L(-1) BOD) was obtained with an estimated detection limit of 4 mg L(-1) BOD. Excellent reproducibility of the BOD sensor was shown with an RSD of 0.9%. Moreover, the BOD sensor showed good tolerance against the presence of copper ions up to a maximum concentration of 0.80 µM (equivalent to 50 ppb). The sensor was applied to BOD measurements of the water from a lake at the University of Indonesia in Jakarta, Indonesia, with results comparable to those made using a standard method for BOD measurement.

Electrochemical detection of tyrosine derivatives and protein tyrosine kinase activity using boron-doped diamond electrodes.

In this report, we determined protein tyrosine kinase (PTKs) activity in human epidermoid carcinoma cells (A431) by employing a novel electrochemical method using boron-doped diamond (BDD) electrodes that enables the electrochemical oxidation of tyrosine (Tyr), phosphorylated Tyr (Tyr-P) and sulfated Tyr (Tyr-S) in water-based solutions. Cyclic voltammetry for Tyr, Tyr-P and Tyr-S showed well-defined oxidation peaks at 0.8 V for Tyr, 1.4V for Tyr-P and 1.7 V for Tyr-S, respectively. Very little work has been reported previously on the detection of Tyr-P and Tyr-S, probably due to their high oxidation potentials. We utilized electrochemical methods for the detection of kinase activity in connection with poly(Glu-Tyr) modified magnetic beads. Linear-sweep voltammograms for the electrochemical detection of PTKs activity were carried out using BDD electrodes consisting of peptide-modified magnetic beads. Without phosphorylation of the peptide-modified magnetic beads using PTKs, we observed clear oxidation peaks for Tyr oxidation and no significant electrochemical responses for Tyr-P oxidation at 1.4V for the background. On the other hand, with phosphorylation of the beads using PTKs, the peak oxidation current at 1.4V clearly increased, while the peak oxidation current for Tyr oxidation decreased. This indicates that PTKs activity could be successfully detected by using electrochemical methods employing BDD electrodes. This method was utilized for the in vitro kinase activity detection of human cell lysate, and the electrochemical measurements were compatible with the Enzyme-Linked ImmunoSorbent Assay based method. Our results indicate that the electrochemical method can be applied to real samples such as cell lysate.

Controlled storage of ferrocene derivatives as redox-active molecules in dendrimers.

Dendritic polyphenylazomethines (DPA) could encapsulate ferroceniums by complexation of the electron-donating skeleton of the DPA imines. Upon addition of ferroceniums to a series of dendritic polyphenylazomethines (DPAGX, where X is the generation number, X = 1-4), the UV-vis spectra showed changes in a manner similar to that observed for the complexation of metal ions with DPAGX. Stepwise shifts in the isosbestic point were consistently observed with the number of imine groups in the first and second layers of the generation-4 dendrimer (DPAG4). DPAG2 and DPAG3 were also found to trap 6 equiv of ferroceniums. To investigate the complexation, UV-vis spectroscopy, (57)Fe Mossbauer spectroscopy, electrospray ionization-mass spectroscopy (ESI-MS), cyclic voltammetry (CV), and fluorescence spectroscopy were performed. We confirmed that neutral ferrocenes cannot complex with the imine group while ferroceniums can. Utilizing the redox property of ferrocenes, we were able to electrochemically control the encapsulation and release of ferrocenes into the DPA in a manner similar to redox-responsive proteins such as ferritin. In addition to ferrocenes, oligoferrocenes could also be trapped in the DPA. The biferrocene cation(1+) was particularly suitable for electrochemical switching due to its stable mixed valence condition. The terferrocene dication(2+) encapsulated into DPAG4 could be fabricated into a thin film, which exhibited the near-infrared absorption of an intervalence charge-transfer (IV-CT) band, pointing the way toward the use of such systems in material science.

An iron(II) incomplete spin-crossover compound: pressure effects and Mössbauer spectroscopy study.

A one-dimensional iron(II) spin-crossover compound [Fe(3py-im)(2)(NCS)(2)].7H(2)O (1) (3py-im = 2,4,5-tris(4-pyridly)-imidazole) has been solvothermally synthesized and structurally characterized. Compound 1 crystallizes in the monoclinic space group P2/c with a = 11.9078(2), b = 9.9474(1), c = 17.7290(3) and beta = 102.361(2) degrees at 105 K. Studies on the variable-temperature magnetic susceptibilities and Mössbauer spectra suggest that compound undergoes incomplete spin transition behaviour. Pressure effects on the transition behaviour have also been investigated, the thermal-induced spin transition becomes more gradual and the critical temperature shifts towards slightly higher temperature range when external pressure increases. However, the spin transition can not be completed by applying external pressure even as high as 0.79 GPa.