Surface Functionalization Strategies of Polystyrene for the Development Peptide-Based Toxin Recognition.

The development of a robust surface functionalization method is indispensable in controlling the efficiency, sensitivity, and stability of a detection system. Polystyrene (PS) has been used as a support material in various biomedical fields. Here, we report various strategies of polystyrene surface functionalization using siloxane derivative, divinyl sulfone, cyanogen bromide, and carbonyl diimidazole for the immobilization of biological recognition elements (peptide developed to detect ochratoxin A) for a binding assay with ochratoxin A (OTA). Our objective is to develop future detection systems that would use polystyrene cuvettes such as immobilization support of biological recognition elements. The goal of this article is to demonstrate the proof of concept of this immobilization support. The results obtained reveal the successful modification of polystyrene surfaces with the coupling agents. Furthermore, the immobilization of biological recognition elements, for the OTA binding assay with horseradish peroxidase conjugated to ochratoxin A (OTA-HRP) also confirms that the characteristics of the functionalized peptide immobilized on polystyrene retains its ability to bind to its ligand. The presented strategies on the functionalization of polystyrene surfaces will offer alternatives to the possibilities of immobilizing biomolecules with excellent order- forming monolayers, due to their robust surface chemistries and validate a proof of concept for the development of highly efficient, sensitive, and stable future biosensors for food or water pollution monitoring.

Electrochemical primer extension based on polyoxometalate electroactive labels for multiplexed detection of single nucleotide polymorphisms.

Polyoxymetalates (POMs) ([SiW11O39{Sn(CH2)2CO)}]4- and [P2W17O61{Sn(CH2)2CO)}]6-) were used to modify dideoxynucleotides (ddNTPs) through amide bond formation, and applied to the multiplexed detection of single nucleotide polymorphisms (SNPs) in an electrochemical primer extension reaction. Each gold electrode of an array was functionalised with a short single stranded thiolated DNA probe, specifically designed to extend with the POM-ddNTP at the SNP site to be interrogated. The system was applied to the simultaneous detection of 4 SNPs within a single stranded 103-mer model target generated using asymmetric PCR, highlighting the potential of POM-ddNTPs for targeted, multiplexed SNP detection. The four DNA bases were successfully labelled with both ([SiW11O39{Sn(CH2)2CO)}]4- and [P2W17O61{Sn(CH2)2CO)}]6-), and [SiW11O39{Sn(CH2)2CO)}]4- demonstrated to be the more suitable due to its single oxidation peak, which provides an unequivocal signal. The POM-ddNTP enzymatically incorporated to the DNA anchored to the surface was visualised by AFM using gold coated mica. The developed assay has been demonstrated to be highly reproducible, simple to carry out and with very low non-specific background signals. Future work will focus on applying the developed platform to the detection of SNPs associated with rifampicin resistance in real samples from patients suffering from tuberculosis.

Stable Carboxylate-Terminated Gold Surfaces Produced by Spontaneous Grafting of an Alkyltin Compound.

Self-assembled monolayers formed by chemisorption of thiolated molecules on gold surfaces are widely applied for biosensing. Moreover, and due to the low stability of thiol-gold chemistry, contributions to the functionalisation of gold substrates with linkers that provide a more stable platform for the immobilisation of electroactive or biological molecules are highly appreciated. Herein, it is demonstrated that a carboxylated organotin compound can be successfully grafted onto gold substrates to form a highly stable organic layer with reactivity for subsequent binding to an aminated molecule. A battery of techniques were used to characterise the surface chemistry. The grafted layer was used to anchor aminoferrocene and subjected to both thermostability tests and long-term stability studies over a period of one year, demonstrating thermostability up to 90 °C and storage stability for at least 12 months at 4 °C protected from light. The stable surface tethering of molecules on gold substrates can be exploited in a plethora of applications, including molecular techniques, such as solid-phase amplification and solid-phase melting curve analysis, that require elevated temperature stability, as well as biosensors, which require long-term storage stability.

PCR Incorporation of Polyoxometalate Modified Deoxynucleotide Triphosphates and Their Application in Molecular Electrochemical Sensing of Yersinia pestis.

Redox-labeled nucleotides are of increasing interest for the fabrication of next generation molecular tools and should meet requirements of being thermally stable, sensitive, and compatible with polymerase-mediated incorporation while also being electrochemically discriminable. The synthesis and characterization of Keggin and Dawson polyoxometalate-deoxynucleotide (POM-dNTP) bioconjugates linked through 7-deaza-modified purines is described. The modified POM-dNTPs were used for polymerase-based amplification of a DNA sequence specific for Yersinia pestis and the amplified DNA detected using an electrochemical DNA sensor. This highlights the potential of polyoxometalates as thermally stable, sensitive and polymerase-compatible redox labels for exploitation in bioanalytical applications.

Biofunctionalization of Polyoxometalates with DNA Primers, Their Use in the Polymerase Chain Reaction (PCR) and Electrochemical Detection of PCR Products.

The bioconjugation of polyoxometalates (POMs), which are inorganic metal oxido clusters, to DNA strands to obtain functional labeled DNA primers and their potential use in electrochemical detection have been investigated. Activated monooxoacylated polyoxotungstates [SiW11 O39 {Sn(CH2 )2 CO}](8-) and [P2 W17 O61 {Sn(CH2 )2 CO}](6-) have been used to link to a 5'-NH2 terminated 21-mer DNA forward primer through amide coupling. The functionalized primer was characterized by using a battery of techniques, including electrophoresis, mass spectrometry, as well as IR and Raman spectroscopy. The functionality of the POM-labeled primers was demonstrated through hybridization with a surface-immobilized probe. Finally, the labeled primers were successfully used in the polymerase chain reaction (PCR) and the PCR products were characterized by using electrophoresis.

Surface functionalisation of carbon for low cost fabrication of highly stable electrochemical DNA sensors.

An alternative strategy for surface tethering of DNA probes, where highly reactive glassy carbon (GC) substrates are prepared via electrochemical hydrogenation and electrochemical/chemical chlorination is reported. Thiolated DNA probes and alkanethiols were stably immobilised on the halogenated carbon, with electrochemical chlorination being milder, thus producing less damage to the surface. Electrochemical DNA sensors prepared using this surface chemistry on carbon with electrochemical chlorination providing an improved performance, producing a highly ordered surface and the use of lateral spacers to improve steric accessibility to immobilised probes was not required.

Facile electrochemical hydrogenation and chlorination of glassy carbon to produce highly reactive and uniform surfaces for stable anchoring of thiolated molecules.

Carbon is a highly adaptable family of materials and is one of the most chemically stable materials known, providing a remarkable platform for the development of tunable molecular interfaces. Herein, we report a two-step process for the electrochemical hydrogenation of glassy carbon followed by either chemical or electrochemical chlorination to provide a highly reactive surface for further functionalization. The carbon surface at each stage of the process is characterized by AFM, SEM, Raman, attenuated total reflectance (ATR) FTIR, X-ray photoelectron spectroscopy (XPS), and electroanalytical techniques. Electrochemical chlorination of hydrogen-terminated surfaces is achieved in just 5 min at room temperature with hydrochloric acid, and chemical chlorination is performed with phosphorus pentachloride at 50 °C over a three-hour period. A more controlled and uniform surface is obtained using the electrochemical approach, as chemical chlorination is observed to damage the glassy carbon surface. A ferrocene-labeled alkylthiol is used as a model system to demonstrate the genericity and potential application of the highly reactive chlorinated surface formed, and the methodology is optimized. This process is then applied to thiolated DNA, and the functionality of the immobilized DNA probe is demonstrated. XPS reveals the covalent bond formed to be a C-S bond. The thermal stability of the thiolated molecules anchored on the glassy carbon is evaluated, and is found to be far superior to that on gold surfaces. This is the first report on the electrochemical hydrogenation and electrochemical chlorination of a glassy carbon surface, and this facile process can be applied to the highly stable functionalization of carbon surfaces with a plethora of diverse molecules, finding widespread applications.