Woo-Seok Choe

Determination of endotoxin through an aptamer-based impedance biosensor.

Lipopolysaccharide (LPS) often referred to endotoxin is an undesirable impurity frequently entrained with various recombinant protein therapeutics and plasmid DNA (pDNA) vaccines of bacterial origin. The inherent toxicities (e.g. fever, hypotension, shock and death) of LPS render its early and sensitive detection essential for several biological assays and/or parenteral administrations of biotherapeutics. In this study, an electrochemical biosensor using an LPS specific single stranded DNA (ssDNA) aptamer as a probe was developed. Amine-terminated aptamer exhibiting high affinity (K(d)=11.9 nM) to LPS was immobilized on a gold electrode using 3-mercaptopropionic acid (MPA) as a linker. Each step of the modification process was characterized by cyclic voltammetry (CV) and electrochemical impendence spectroscopy (EIS). A good linear relationship of the changes in the charge-transfer resistance (ΔR(et)) and the logarithmic value of LPS concentration was demonstrated in a broad dynamic detection range of 0.001-1 ng/ml. Furthermore, the aptasensor showed a high selectivity to LPS despite the presence of pDNA, RNA and bovine serum albumin (BSA) and could be regenerated in low pH condition, offering a promising option for detecting LPS often present in a complex milieu.

Electrochemical analysis of copper ion using a Gly-Gly-His tripeptide modified poly(3-thiopheneacetic acid) biosensor.

A novel biosensor harnessing a conducting polymer functionalized with a copper ion specific peptide proved to be highly effective for electrochemical analysis of copper ions. The developed sensor comprised a transducer based on a conducting polymer (poly(3-thiopheneacetic acid)) electrode and a probe (tripeptide, Gly-Gly-His) selectively cognitive of copper ions. For functionalization of the electrode, the carboxylic group of the polymer was covalently coupled with the amine group of the tripeptide, and its structural features were confirmed by X-ray photoelectron spectroscopy (XPS) and attenuated total reflection infrared (ATR-IR) spectroscopy. The peptide modified polythiophene biosensor was used for the electrochemical analysis of various trace metal ions by square wave voltammetry. The electrode was found to be highly sensitive and selective to Cu(2+) in the range of 0.02-20 microM with almost no cross binding to other metal ions such as Ni(2+) and Pb(2+). Furthermore, the developed sensor exhibited a high stability and reproducibility despite the repeated use of the sensor electrode and probe. With the advent of more diverse affinity bioprobes specific towards a broad range of analytes, the demonstrated strategy harnessing peptide modified polythiophene biosensor is likely to provide an excellent platform for the selective determination of trace amount of analytes whose detection is otherwise cumbersome.

Harnessing aptamers for electrochemical detection of endotoxin.

Lipopolysaccharide (LPS), also known as endotoxin, triggers a fatal septic shock; therefore, fast and accurate detection of LPS from a complex milieu is of primary importance. Several LPS affinity binders have been reported so far but few of them have proved their efficacy in developing electrochemical sensors capable of selectively detecting LPS from crude biological liquors. In this study, we identified 10 different single-stranded DNA aptamers showing specific affinity to LPS with dissociation constants (K(d)) in the nanomolar range using a NECEEM-based non-SELEX method. Based on the sequence and secondary structure analysis of the LPS binding aptamers, an aptamer exhibiting the highest affinity to LPS (i.e., B2) was selected to construct an impedance biosensor on a gold surface. The developed electrochemical aptasensor showed excellent sensitivity and specificity in the linear detection range from 0.01 to 1 ng/mL of LPS with significantly reduced detection time compared with the traditional Limulus amoebocyte lysate (LAL) assay.

Polypyrrole nanowire modified with Gly-Gly-His tripeptide for electrochemical detection of copper ion

We developed a novel biosensor comprising a transducer of carboxyl end-capped overoxidized polypyrrole nanowire electrode and a probe of tripeptide (Gly-Gly-His) selectively cognitive of Cu2+. The developed sensor was demonstrated to specifically detect Cu2+ in the nanomolar range. The diameter of the polypyrrole nanowires, prepared by one step template-free polymerization, was approximately 60-90 nm. For functionalization of the electrode, the carboxyl group was introduced by the addition of pyrrole-α-carboxylic acid which was covalently coupled with the amine group of the tripeptide. The structural features of the peptide functionalized nanowire electrode were confirmed by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) spectroscopy. Using cyclic and square wave voltammetry, the nanowire biosensor proved to be highly sensitive to Cu2+ in the range of 20-300 nM.

Nanomesh‐Structured Ultrathin Membranes Harnessing the Unidirectional Alignment of Viruses on a Graphene‐Oxide Film

DOI: 10.1002/adma.201305862 However, strong van der Waals interactions and subsequent irreversible aggregation of the nanomaterials makes it unlikely for the assembled system to exhibit unidirectional alignment. This limitation can be overcome by employing “intelligent” (i.e., responsive) and fl exible one-dimensionally structured biomaterials. [ 11,12 ] The self-assembled structures of M13 viruses are an example; their mechanical stiffness and structural characteristics are readily controlled by manipulating the environmental pH or the type of bonding with the underlying substrate. [ 13,14 ]

Highly sensitive electrochemical lead ion sensor harnessing peptide probe molecules on porous gold electrodes.

Lead ion is one of the most hazardous and ubiquitous heavy metal pollutants and poses an increasing threat to the environment and human health. This necessitates rapid and selective detection and/or removal of lead ions from various soil and water resources. Recently, we identified several Pb²⁺ binding peptides via phage display technique coupled with chromatographic biopanning (Nian et al., 2010) where a heptapeptide (TNTLSNN) capable of recognizing Pb²⁺ with high affinity and specificity evolved. In the present study, an electrochemical sensor harnessing this Pb²⁺ affinity peptide as a probe on a porous gold electrode was developed. The three dimensional porous gold electrode was obtained from electrochemical deposition using the dynamic hydrogen bubble template method. A thin layer of poly(thiophene acetic acid) (PTAA) was coated on the porous gold surface. The Pb²⁺ recognizing peptide was immobilized via amide linkage on the PTAA. The developed biosensor was demonstrated to be fast, selective and reproducible in Pb²⁺ etection, exhibiting Pb²⁺-specific peak current values around -0.15 V in a broad concentration range (1-1×10⁷ nM) in 10 min despite the repeated use after regeneration.

Highly sensitive and selective determination of bisphenol-A using peptide-modified gold electrode.

Fast and accurate determination of bisphenol A (BPA) in varying matrices has become important in recent years. In this study, a cysteine-flanked heptapeptide sequence Cys-Lys-Ser-Leu-Glu-Asn-Ser-Tyr-Cys (CKSLENSYC), which is capable of recognizing BPA with high specificity, was isolated using a phage display technique. A novel electrochemical biosensor harnessing this affinity peptide as a BPA detection probe, was constructed and its performance was assessed. The formation of a self-assembled peptide monolayer on the gold electrode was confirmed by attenuated total reflection infrared spectroscopy (ATR-IR), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Following the exploration of the optimum sensing condition, differential pulse voltammetry (DPV) was used to determine the varying concentrations of BPA in the solution. The developed sensor conveyed excellent performance in view of sensing speed, sensitivity and selectivity by detecting BPA in less than 5 min with a broad dynamic detection range of 1-5000 nM of BPA, despite the presence of several interfering species, such as phenolic compounds and inorganic ions.

Highly Sensitive Reduced Graphene Oxide Impedance Sensor Harnessing π‑Stacking Interaction Mediated Direct Deposition of Protein Probes

Graphene-based electrochemical impedance sensors have recently received much attention due to their outstanding sensing capability and economic viability. In this study, we present a novel means of constructing an impedance sensing platform via harnessing intrinsic π-stacking interactions between probe protein molecules and reduced graphene oxide (RGO) substrate, obviating the need for introducing external chemical groups often required for covalent anchoring of the probes. To achieve this goal, protein molecules used as a probe were denatured to render their hydrophobic residues exposed in order to facilitate their direct π-stacking interactions with the surface of RGO nanosheets. The protein molecules in denatured form, which would otherwise have difficulty in undergoing π-stacking interactions with the RGO surface, were found to uniformly cover the RGO nanosheets at high density, conducive to providing a graphene-based impedance sensing platform capable of detecting a probe-specific analyte at high sensitivity. The proof-of-concept performance of thus-constructed RGO-based impedance sensors was demonstrated via selective detection of biological binding events of antigen-antibody reaction at a femtomolar range. Notably, since the π-stacking interaction can occur on the entire RGO surface, it can desirably exclude a backfill process indispensable for the conventional biosensors to suppress background noise signals. Since the procedure of π-stacking mediated direct deposition of on-purpose denatured protein probes onto the RGO surface is facile and straightforward, the proposed strategy is anticipated to extend its applicability for fabrication of high performance graphene-based bio or chemical sensors.

Selective detection of endotoxin using an impedance aptasensor with electrochemically deposited gold nanoparticles

Using a single-stranded DNA (ssDNA) aptamer exhibiting high binding affinity (Kd = 12 nM) to endotoxin as a probe, an impedance sensor where aptamer-conjugated gold nanoparticles (AuNPs) were electrochemically deposited on a gold electrode was fabricated and its performance in regard to endotoxin detection assessed. AuNPs have been employed widely as biosensors because of their unique physical and chemical properties. In order to maximize the performance of the impedance aptasensor on endotoxin detection, some critical factors affecting aptamer conjugation to AuNPs and target recognition ability (i.e. concentrations of aptamer coupled with AuNPs, pH, ion strength and cation effect at the time of aptamer–endotoxin interaction) were optimized. Electrochemical impendence spectroscopy, cyclic voltametry, atomic force microscope, scanning electron microscope and quartz crystal microbalance were employed to characterize all the modification/detection procedures during the sensor fabrication. The developed aptasensor showed a broad linear dynamic detection range (0.01–10.24 ng/ml) with a very low detection limit for endotoxin (0.005 ng/ml), despite the presence of several biomolecules (e.g. plasmid DNA, RNA, serum albumin, Glc and sucrose) known to interfere with other endotoxin assays. The demonstrated aptasensor required a detection time of only 10 min, providing a simple and fast analytical method to specifically detect endotoxin from complex biological liqors.

Affinity analysis of DNA aptamer-peptide interactions using gold nanoparticles.

Gold nanoparticles (AuNPs) were used as colorimetric probe and fluorescence quencher for affinity analysis of DNA aptamers toward their target mucin 1 (MUC1) peptide. Single-stranded DNA (ssDNA) aptamer-coated AuNPs showed increased stability (i.e., more resistant to aggregation induced by NaCl) in the presence of their target peptide due to the increase in steric protection conferred by the ssDNA-peptide complexes formed on the AuNPs. Based on changes in the UV-vis extinction spectrum of AuNPs (a measure of AuNPs aggregation) and fluorescence restoration of CY5-ssDNA upon ssDNA-peptide complex formation, the formation of the complexes and ssDNA sequence-dependent dissociation constant (K(d)) were determined. Besides the UV-vis and fluorescence measurements, the hydrodynamic diameters, zeta potential measurements, and transmission electron microscopy (TEM) images of AuNPs after various coatings supported the assay principle. The methodology presented herein provides a rapid and sensitive alternative solution for the identification of high affinity binders from systematic evolution of ligands by exponential enrichment (SELEX).