Interpretation of Protein Adsorption through Its Intrinsic Electric Charges: A Comparative Study Using a Field-Effect Transistor, Surface Plasmon Resonance, and Quartz Crystal Microbalance

We describe the highly sensitive detection of the nonspecific adsorption of proteins onto a 1-undecanethiol self-assembled monolayer (SAM)-formed gold electrode by parallel analysis using field effect transistor (FET), surface plasmon resonance (SPR), and quartz crystal microbalance (QCM) sensors. The FET sensor detects the innate electric charges of the adsorbed protein at the electrode/solution interface, transforming the change in charge density into a potentiometric signal in real time, without the requirement for labels. In particular, using the Debye–Huckel model, the degree of potential shift was proportional to the dry mass of adsorbed albumin and β-casein. A comparison of the FET signal with SPR and QCM data provided information on the conformation and orientation of the surface-bound protein by observing characteristic break points in the correlation slopes between the signals. These slope transitions reflect a multistage process that occurs upon protein adsorption as a function of protein concentration, including interim coverage, film dehydration, and monolayer condensation. The FET biosensor, in combination with SPR and QCM, represents a new technology for interrogating protein–material interactions both quantitatively and qualitatively

AlCl₃‑Mediated Aldol Cyclocondensation of 1,6- and 1,7-Diones to Cyclopentene and Cyclohexene Derivatives

Exactly 1/3 mol of AlCl₃ is sufficient to cyclize 1 mol of 1,ω-dibenzoylbutane (or pentane) to a cyclopentenone (or hexenone) derivative in high yield at room temperature in 40 min to several hours. This condensation is driven by removing elements of water as HCl and Al­(OH)₃, and the product enones are exclusively unconjugated, unlike the base-catalyzed condensations providing thermodynamically more stable conjugated enones

Simultaneous Monitoring of Protein Adsorption Kinetics Using a Quartz Crystal Microbalance and Field-Effect Transistor Integrated Device

We developed an integrated device comprising a quartz crystal microbalance (QCM) and a field-effect transistor (FET) with a single common gold electrode in a flow chamber. An alternating current inducing oscillations in the piezoelectric quartz of the QCM sensor is electrically independent of the circuit for the FET output so that the two sensors in different detection mechanisms simultaneously record binding kinetics from a single protein solution on the same electrode. A conjunction of adsorbed mass from QCM with electric nature of bound protein from FET provided deeper understanding on a complex process of nonspecific protein adsorption and subsequent conformational changes at a solid/liquid interface. Lower apparent <i>k</i>_on values obtained by FET than those obtained by QCM on hydrophobic surfaces are interpreted as preferred binding of protein molecules facing uncharged domains to the electrode surface, whereas higher <i>k</i>_off values by FET than those by QCM imply active macromolecular rearrangements on the surfaces mainly driven by hydrophobic association in an aqueous medium. The advanced features of the combined sensor including in situ, label-free, and real-time monitoring provide information on structural dynamics, beyond measurements of affinities and kinetics in biological binding reactions

Sialic acid biosensing by post-printing modification of PEDOT:PSS with pyridylboronic acid

A poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS)-based conducting polymer, which has biorecognition capabilities, has promising biosensing applications. Previously, we developed a facile method for post-printing chemical modification of PEDOT:PSS thin films from commercial sources. Molecular recognition elements were directly introduced into the PSS side chain by a two-step chemical reaction: introduction of an ethylenediamine linker via an acid chloride reaction of the sulfonate moiety, and subsequent receptor attachment to the linker via amine coupling. In this study, the same method was used to introduce 6-carboxypyridine-3-boronic acid (carboxy-PyBA) into the linker for specifically detecting N-acetylneuraminic acid (sialic acid, SA), as a cancer biomarker. The surface-modified PEDOT:PSS films were characterized by X-ray photoelectron spectroscopy, attenuated total reflection Fourier-transform infrared spectroscopy, and static water contact angle and conductivity measurements. The specific interaction between PyBA and SA was detected by label-free reagent-free potentiometry. The SA-specific negative potential responses of modified PEDOT:PSS electrodes, which was ascribed to an SA carboxyl anion, were observed in a physiologically relevant SA range (1.6–2.9 mM) at pH 5, in a concentration-dependent manner even in the presence of 10% fetal bovine serum. The sensitivity was −2.9 mV/mM in 1–5 mM SA with a limit of detection of 0.7 mM. The sensing performances were almost equivalent to those of existing graphene-based electrical SA sensors. These results show that our chemical derivatization method for printing PEDOT:PSS thin films will have applications in SA clinical diagnostics.</p

Boronate Based Metal-Free Platform for Diphosphate-Specific Molecular Recognitions

A reversible boronate–diol interaction provides a versatile synthetic platform for molecular recognitions whose binding specificity can be molecularly tailored. We found that boronate derivatives with relatively strong acidity generally undergo a diphosphate-specific recognition among other phosphates under weakly acidic pH conditions, a feature relevant to DNA sequencing. ¹¹B and ³¹P NMR studies identified “tetrahedral boronate and divalent diphosphate” as a pair responsible for forming a 1:1 stoichiometric complex, which manifests as a unique pH-dependent stability

Investigation of the Mechanism of Adsorption of β-Nicotinamide Adenine Dinucleotide on Single-Walled Carbon Nanotubes

We address in this manuscript the important issue of the stability of single-walled carbon nanotube (SWCNT)-based electrodes upon oxidation of NADH to NAD⁺. NADH and NAD⁺ play a key role in the development of electrochemical enzyme-based biofuel cells and biosensors. However, most of the electrode materials exhibit strong surface passivation when oxidation of NADH to NAD⁺ occurs. SWCNT-based electrodes are not affected by such a passivation effect. In the present work, we address the fundamental question, “why are the single-walled carbon nanotube electrodes prone to passivation?” using electrochemical methods and first-principles molecular dynamics simulations. We found that this is due to the wide exposed surface of SWCNT-based electrodes rather than other “inherent” properties of SWCNTs, such as the electrocatalytic effect and high curvature

Design of the microdevice.

<p>a) Schematic view of the cross-section of the device (not drawn to scale). The height of the microperfusion channel is around 200 µm. A typical Xenopus oocyte would be 1000–1200 µm in diameter. b) Micrograph of the sensor as seen through the hole (ca. 800 µm in diameter) of the oocyte immobilization compartment. The oocyte membrane completely covers the active area of the sensor due to its deformability. c) Exploded view of the device using the original 3D CAD engineering data. d) Photograph of the assembled, but unconnected device.</p

Specific Recognition of Human Influenza Virus with PEDOT Bearing Sialic Acid-Terminated Trisaccharides

Conducting polymers are good candidates for biosensor applications when molecular recognition element is imparted. We developed trisaccharide-grafted conducting polymers for label-free detection of the human influenza A virus (H1N1) with high sensitivity and specificity. A 3,4-ethylenedioxythiophene (EDOT) derivative bearing an oxylamine moiety was electrochemically copolymerized with EDOT. The obtained film was characterized by cyclic voltammetry, X-ray photoelectron spectroscopy, scanning electron microscopy, stylus surface profilometer, and AC-impedance spectroscopy. The trisaccharides comprising Sia-α2,6′-Gal-Glu (2,6-sialyllactose) or Sia-α2,3′-Gal-Glu (2,3-sialyllactose) were covalently introduced to the side chain of the conducting polymers as a ligand for viral recognition. Immobilization of sialyllactose was confirmed by quartz crystal microbalance (QCM) and water contact angle measurements. Specific interaction of 2,6-sialyllactose with hemagglutinin in the envelope of the human influenza A virus (H1N1) was detected by QCM and potentiometry with enhanced sensitivity by 2 orders of magnitude when compared with that of commercially available kits. The developed conducting polymers possessing specific virus recognition are a good candidate material for wearable monitoring and point-of-care testing because of their processability and mass productivity in combination with printing technologies

Membrane transport experiments.

<p>Experiments conducted on oocytes heterologously expressing various membrane transport proteins indicated with their respective controls on non-injected (NI) oocytes showing sensor readout (V_SG) as a function of time. Only part of the initial stabilizing baseline region that preceded substrate application is shown (see Materials and Methods): a) PAT1, b) NaPi IIb, c) NaPi-IIc, d) PiT-2, e) Proline control, f) P_i control, g) GAT1, h) ENaC. In each case either the same or representative oocytes from the same batch were pretested using a two-electrode voltage clamp to confirm functional expression. The bars indicate the duration of application of the respective activating and blocking agents. Arrows indicate flux direction of substrate according to the assumed driving force conditions.</p

Correlation of pH response with protein expression level.

<p>Correlating sensor response with transport activity. a) Sensor response to proline superfusion of a representative oocyte (designated #4 in c) heterologously expressing PAT1. b) TEVC I-V data of the proline-dependent current of oocyte #4 in response to the addition of 3 mM proline solution to the 100 Na buffer. Inset shows the change in membrane potential induced by proline application for the same oocyte as in a. c) Correlation of ΔV_SG and the substrate-dependent current. Each point represents data from a single oocyte. Arrow marks the data point of oocyte #4 (−23 mV, −140 nA).</p