Design the RNA aptamer of PCA3 long non-coding ribonucleic acid by the coarse-grained molecular mechanics.

The stochastic tunneling-basin hopping-discrete molecular dynamics (STUN-BH-DMD) method was applied to predict the tertiary structure of the prostate cancer marker PCA3 using two respective secondary structures predicted by the Vienna RNA package and Mathews lab package. The RNA CG force field with the geometrical restraints for maintaining PCA3 secondary structures is used. For each secondary structure, 5000 PCA3 structures were predicted by using 5000 independent initial structures. These structures were then evaluated by a scoring function, considering the contributions from the radius of gyration, contact energy, and surface fraction of complementary nucleotides to ASO683 and ASO735 used in the related experiment. For each secondary structure, the PCA3 structures with the highest three scores were selected for aptamer design and further adsorption simulation. The ASOs complementary to PCA3 surface segments possessing relatively higher RMSF values are selected to be the potential PCA3 aptamers. After the adsorption simulation, the adsorption energies of ASO961, ASO3181, ASO3533, and ASO3595 are higher than or comparable to those of ASO683 and ASO735 used in the experiment. The NEB method was used to obtain MEPs for the adsorption process of all predicted ASOs onto PCA3. The adsorption barriers range between 29 ∼ 39 kcal/mol, while the desorption barriers range between 112 ∼ 352 kcal/mol, indicating these aptamer/PCA3 complexes are very stable. Using PCA3 surface segments with relatively higher RMSF values, longer ASOs can be also obtained and most longer ASOs possess lower binding energy, ranging between -486.1 and -618.2 kcal/mol.Communicated by Ramaswamy H. Sarma.

Exploring the most stable aptamer/target molecule complex by the stochastic tunnelling-basin hopping-discrete molecular dynamics method.

The stochastic tunnelling-basin hopping-discrete molecular dynamics (STUN-BH-DMD) method was applied to the search for the most stable biomolecular complexes in water by using the MARTINI coarse-grained (CG) model. The epithelial cell adhesion molecule (EpCAM, PDB code: 4MZV) was used as an EpCAM adaptor for an EpA (AptEpA) benchmark target molecule. The effects of two adsorption positions on the EpCAM were analysed, and it is found that the AptEpA adsorption configuration located within the EpCAM pocket-like structure is more stable and the energy barrier is lower due to the interaction with water. By the root mean square deviation (RMSD), the configuration of EpCAM in water is more conservative when the AptEpA binds to EpCAM by attaching to the pocket space of the EpCAM dimer. For AptEpA, the root mean square fluctuation (RMSF) analysis result indicates Nucleobase 1 and Nucleobase 2 display higher flexibility during the CGMD simulation. Finally, from the binding energy contour maps and histogram plots of EpCAM and each AptEpA nucleobase, it is clear that the binding energy adsorbed to the pocket-like structure is more continuous than that energy not adsorbed to the pocket-like structure. This study has proposed a new numerical process for applying the STUN-BH-DMD with the CG model, which can reduce computational details and directly find a more stable AptEpA/EpCAM complex in water.

Development of amperometric α-ketoglutarate biosensor based on ruthenium-rhodium modified carbon fiber enzyme microelectrode.

A rapid and highly sensitive miniaturized amperometric biosensor for the detection of α-ketoglutarate (α-KG) based on a carbon fiber electrode (CFE) is presented. The biosensor is constructed by immobilizing the enzyme, glutamate dehydrogenase (GLUD) on the surface of single carbon fiber modified by co-deposition of ruthenium (Ru) and rhodium (Rh) nanoparticles. SEM and EDX shed useful insights into the morphology and composition of the modified microelectrode. The mixed Ru/Rh coating offers a greatly enhanced electrocatalytic activity towards the detection of ß-nicotinamide adenine dinucleotide (NADH), with a substantial decrease in overpotential of ∼ 400 mV compared to the unmodified CFE. It also imparts higher stability with minimal surface fouling, common to NADH oxidation. Further modification with the enzyme, GLUD leads to effective amperometric biosensing of α-KG through monitoring of the NADH consumption. A very rapid response to dynamic changes in the α-KG concentrations is observed with a response time of 6s. The current response is linear between 100 and 600 µM with a sensitivity of 42 µAM(-1) and a detection limit of 20 µM. This proof of concept study indicates that the GLUD-Ru/Rh-CFE biosensor holds great promise for real-time electrochemical measurements of α-KG.

Strip-based amperometric detection of myeloperoxidase.

The development of a screen-printed strip-based amperometric biosensor for the determination of myeloperoxidase (MPO) levels is reported. The biosensor utilizes 3,3',5,5'-tetramethylbenzidine (TMB) as a redox mediator to enable high-sensitivity quantification of physiological levels of MPO. A multivariate parameter optimization was performed. Under the optimal conditions, physiological levels of MPO between 3 and 18 U/L were detected in both acetate buffer (pH 4.5) and human serum using flexible screen-printed electrodes (SPE). The potential interference generated by common serum-based electroactive compounds and a similar peroxidase enzyme was also investigated. The proposed detection methodology offers a simpler, more rapid, and cost-effective alternative to conventional MPO immunoassays, thereby leading to further development in point-of-care testing of acute cardiac events.

Flexible thick-film glucose biosensor: influence of mechanical bending on the performance.

The influence of the bending-induced mechanical stress of flexible Nafion/GOx/carbon screen-printed electrodes (SPEs) upon the performance of such glucose biosensors has been examined. Surprisingly, such flexible enzyme/polymer-SPEs operate well following a severe bending-induced mechanical stress (including a 180 degrees pinch), and actually display a substantial sensitivity enhancement following their mechanical bending. The bending-induced sensitivity enhancement is observed only for the amperometric detection of the glucose substrate but not for measurements of hydrogen peroxide, catechol or ferrocyanide at coated or bare SPEs. These (and additional) data indicate that the bending effect is associated primarily with changes in the biocatalytic activity. Such sensitivity enhancement is more pronounced at elevated glucose levels, reflecting the bending-induced changes in the biocatalytic reaction. Factors affecting the bending-induced changes in the performance are examined. While our data clearly indicate that flexible enzyme/polymer-SPEs can tolerate a severe mechanical stress and hold promise as wearable glucose biosensors, delivering the sample to the active sensor surface remains the major challenge for such continuous health monitoring.

Effects of electric potential treatment of a chromium hexacyanoferrate modified biosensor based on PQQ-dependent glucose dehydrogenase.

A novel potential treatment technique applied to a glucose biosensor that is based on pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) and chromium hexacyanoferrate (CrHCF) incorporated into a platinum (Pt) electrode was demonstrated. CrHCF, serving as a mediator, was electrochemically deposited on the Pt electrode as ascertained by CV, SEM, FTIR and XPS measurements. The potential treatment of CrHCF, which converts Fe(II) to Fe(III), enables the glucose detection. The amperometric measurement linearity of the biosensor was up to 20 mM (R = 0.9923), and the detection sensitivity was 199.94 nA/mM per cm(2). More importantly, this biosensor remained stable for >270 days.

Thermally Stable Improved First-Generation Glucose Biosensors based on Nafion/Glucose-Oxidase Modified Heated Electrodes.

We illustrate how the use of heated electrodes enhances the performance of glucose biosensors based on amperometric detection of the glucose-oxidase generated hydrogen peroxide. Nafion is shown to be an excellent matrix to protect glucose oxidase from thermal inactivation during the heating pulses. The influence of the electrode temperature upon the amperometric response is examined. Temperature pulse amperometry (TPA) has been used to obtain convenient peak-shaped analytical signals. Surprisingly, up to 67.5 °C, the activity of Nafion-entrapped glucose oxidase is greatly enhanced (24 -fold) by accelerated kinetics rather than decreased by thermal inactivation. Amperometric signals even at elevated temperatures are stable upon prolonged operation involving repetitive measurements. The linear calibration range is significantly extended.

A miniaturized glucose biosensor for in vitro and in vivo studies.

A miniaturized wireless glucose biosensor has been developed to perform in vitro and in vivo studies. It consists of an external control subsystem and an implant sensing subsystem. The implant subsystem consists of a micro-processor, which coordinates circuitries of radio frequency, power regulator, command demodulator, glucose sensing trigger and signal read-out. Except for a set of sensing electrodes, the micro-processor, the circuitries and a receiving coil were hermetically sealed with polydimethylsiloxane. The electrode set is a substrate of silicon oxide coated with platinum, which includes a working electrode and a reference electrode. Glucose oxidase was immobilized on the surface of the working electrode. The implant subsystem bi-directionally communicates with the external subsystem via radio frequency technologies. The external subsystem wirelessly supplies electricity to power the implant, issues commands to the implant to perform tasks, receives the glucose responses detected by the electrode, and relays the response signals to a computer through a RS-232 connection. Studies of in vitro and in vivo were performed to evaluate the biosensor. The linear response of the biosensor is up to 15 mM of glucose in vitro. The results of in vivo study show significant glucose variations measured from the interstitial tissue fluid of a diabetes rat in fasting and non-fasting periods.

Using MPTMS as permselective membranes of biosensors.

A sol-gel material of (3-mercaptopropyl) trimethoxysilane (MPTMS) is proposed to function as permselective membranes of biosensors. Permselectivity of MPTMS and Nafion was compared by studying their anti-interferent ability. Membrane porosity of MPTMS and Nafion was first confirmed via voltammetric responses in ferrocynite/ferricynite solution. In the comparison studies, membranes prepared with 20% MPTMS in phosphate buffer solution (PBS) and 1% Nafion in 2-propanol (IPA) were used as coating materials on the surface of two platinum (Pt) electrodes. These electrodes were used to electrochemically measure the response currents of ascorbic acid, uric acid, and acetaminophen. The results indicate that the MPTMS-based electrode produced much less response currents from the interference species compared to that of the Nafion-based electrode. This denotes that the anti-interferent ability ofMPTMS is superior to that of Nafion. A platinum working electrode containing glucose oxidase (GOx) immobilized by poly-aniline (PA) and then modified by MPTMS was developed and evaluated. The results show that the optimum applied potential for the glucose biosensor is 0.4 V. This operational potential not only inhibits the response currents from ascorbic acid, uric acid, and acetaminophen but also produces rather high signals for glucose.

Chromium hexacyanoferrate modified biosensor based on PQQ-dependent glucose dehydrogenase.

A Pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase based platinum electrode was developed to detect glucose. Chromium hexacyanoferrate was modified onto this electrode to serve as an electron transfer mediator between PQQ and the platinum electrode. This biosensor showed the optimal response of glucose measurements at pH 7 and an operation potential of +0.4 V (vs. Ag/AgCl). More importantly the lifespan of the biosensor has been last for 47 days without significant enzyme activity degradation.