27 research outputs found
Chemically Induced pH Perturbations for Analyzing Biological Barriers Using Ion-Sensitive Field-Effect Transistors
Potentiometric pH measurements have long been used for the bioanalysis of biofluids, tissues, and cells. A glass pH electrode and ion-sensitive field-effect transistor (ISFET) can measure the time course of pH changes in a microenvironment as a result of physiological and biological activities. However, the signal interpretation of passive pH sensing is difficult because many biological activities influence the spatiotemporal distribution of pH in the microenvironment. Moreover, time course measurement suffers from stability because of gradual drifts in signaling. To address these issues, an active method of pH sensing was developed for the analysis of the cell barrier in vitro. The microenvironmental pH is temporarily perturbed by introducing a low concentration of weak acid (NH4+) or base (CH3COO−) to cells cultured on the gate insulator of ISFET using a superfusion system. Considering the pH perturbation originates from the semi-permeability of lipid bilayer plasma membranes, induced proton dynamics are used for analyzing the biomembrane barriers against ions and hydrated species following interaction with exogenous reagents. The unique feature of the method is the sensitivity to the formation of transmembrane pores as small as a proton (H+), enabling the analysis of cell–nanomaterial interactions at the molecular level. The new modality of cell analysis using ISFET is expected to be applied to nanomedicine, drug screening, and tissue engineering
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
Oleyl group-functionalized insulating gate transistors for measuring extracellular pH of floating cells
The extracellular ionic microenvironment has a close relationship to biological activities such as by cellular respiration, cancer development, and immune response. A system composed of ion-sensitive field-effect transistors (ISFET), cells, and program-controlled fluidics has enabled the acquisition of real-time information about the integrity of the cell membrane via pH measurement. Here we aimed to extend this system toward floating cells such as T lymphocytes for investigating complement activation and pharmacokinetics through alternations in the plasma membrane integrity. We functionalized the surface of tantalum oxide gate insulator of ISFET with oleyl-tethered phosphonic acid for interacting with the plasma membranes of floating cells without affecting the cell signaling. The surface modification was characterized by X-ray photoelectron spectroscopy and water contact angle measurements. The Nernst response of −37.8 mV/pH was obtained for the surface-modified ISFET at 37 °C. The oleyl group-functionalized gate insulator successfully captured Jurkat T cells in a fluidic condition without acute cytotoxicity. The system was able to record the time course of pH changes at the cells/ISFET interface during the process of instant addition and withdrawal of ammonium chloride. Further, the plasma membrane injury of floating cells after exposure by detergent Triton™ X-100 was successfully determined using the modified ISFET with enhanced sensitivity as compared with conventional hemolysis assays
Measurement of Rapid Amiloride-Dependent pH Changes at the Cell Surface Using a Proton-Sensitive Field-Effect Transistor
We present a novel method for the rapid measurement of pH fluxes at close proximity to the surface of the plasma membrane in mammalian cells using an ion-sensitive field-effect transistor (ISFET). In conjuction with an efficient continuous superfusion system, the ISFET sensor was capable of recording rapid changes in pH at the cells’ surface induced by intervals of ammonia loading and unloading, even when using highly buffered solutions. Furthermore, the system was able to isolate physiologically relevant signals by not only detecting the transients caused by ammonia loading and unloading, but display steady-state signals as would be expected by a proton transport-mediated influence on the extracellular proton-gradient. Proof of concept was demonstrated through the use of 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a small molecule inhibitor of sodium/hydrogen exchangers (NHE). As the primary transporter responsible for proton balance during cellular regulation of pH, non-electrogenic NHE transport is notoriously difficult to detect with traditional methods. Using the NHE positive cell lines, Chinese hamster ovary (CHO) cells and NHE3-reconstituted mouse skin fibroblasts (MSF), the sensor exhibited a significant response to EIPA inhibition, whereas NHE-deficient MSF cells were unaffected by application of the inhibitor
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><sub>on</sub> 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><sub>off</sub> 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
Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes
sensor