Nonaqueous Oxidation in DNA Microarray Synthesis Improves the Oligonucleotide Quality and Preserves Surface Integrity on Gold and Indium Tin Oxide Substrates.

Nucleic acids attached to electrically conductive surfaces are very frequently used platforms for sensing and analyte detection as well as for imaging. Synthesizing DNA on these uncommon substrates and preserving the conductive layer is challenging as this coating tends to be damaged by the repeated use of iodine and water, which is the standard oxidizing medium following phosphoramidite coupling. Here, we thoroughly investigate the use of camphorsulfonyl oxaziridine (CSO), a nonaqueous alternative to I2/H2O, for the synthesis of DNA microarrays in situ. We find that CSO performs equally well in producing high hybridization signals on glass microscope slides, and CSO also protects the conductive layer on gold and indium tin oxide (ITO)-coated slides. DNA synthesis on conductive substrates with CSO oxidation yields microarrays of quality approaching that of conventional glass with intact physicochemical properties.

Dipodal Silanes Greatly Stabilize Glass Surface Functionalization for DNA Microarray Synthesis and High-Throughput Biological Assays.

Glass is by far the most common substrate for biomolecular arrays, including high-throughput sequencing flow cells and microarrays. The native glass hydroxyl surface is modified by using silane chemistry to provide appropriate functional groups and reactivities for either in situ synthesis or surface immobilization of biologically or chemically synthesized biomolecules. These arrays, typically of oligonucleotides or peptides, are then subjected to long incubation times in warm aqueous buffers prior to fluorescence readout. Under these conditions, the siloxy bonds to the glass are susceptible to hydrolysis, resulting in significant loss of biomolecules and concomitant loss of signal from the assay. Here, we demonstrate that functionalization of glass surfaces with dipodal silanes results in greatly improved stability compared to equivalent functionalization with standard monopodal silanes. Using photolithographic in situ synthesis of DNA, we show that dipodal silanes are compatible with phosphoramidite chemistry and that hybridization performed on the resulting arrays provides greatly improved signal and signal-to-noise ratios compared with surfaces functionalized with monopodal silanes.

Determination of p53 biomarker using an electrochemical immunoassay based on layer-by-layer films with NiFe2O4 nanoparticles.

A disposable electrochemical immunosensors is presented suitable to detect cancer biomarker p53 using screen-printed carbon electrodes modified with a layer-by-layer (LbL) matrix of carboxylated NiFe2O4 nanoparticles and polyethyleneimine, onto which anti-p53 antibodies were adsorbed. Under optimized conditions, the immunosensors exhibited high surface coverage and high concentration of immobilized antibodies, which allowed for detection of p53 in a wide dynamic range from 1.0 to 10 × 103 pg mL-1, with a limit of detection of 5.0 fg mL-1 at a working potential of 100 mV vs. Ag/AgCl. The immunosensors also exhibited good selectivity with negligible interference upon incubation in complex matrices containing high concentrations of proteins (i.e., fetal bovine serum and cell lysate). The immunosensor performance is among the best reported in the literature for determination of p53, with the additional advantage of being disposable and operating with low-volume solutions.Graphical abstract Schematic representation of immunosensor fabrication depicting the immobilization of specific antibodies against p53 protein onto the surfaces of disposable printed electrodes modified with films of polyethyleneimine and different concentrations of carboxylated magnetic nanoparticles.

Screen-printed electrodes modified with carbon black and polyelectrolyte films for determination of cancer marker carbohydrate antigen 19-9.

Electrochemical immunosensors have been developed to determine the carbohydrate antigen 19-9 (CA19-9). They are based on screen-printed carbon electrodes (SPCEs) coated with layer-by-layer (LbL) films of carbon black (CB) and polyelectrolytes. Owing to a suitable choice of LbL film architecture, the procedures for immobilization of anti-CA19-9 antibodies on the electrode surfaces were straightforward. Mechanically flexible immunosensors were capable of detecting CA19-9 within a dynamic range of 0.01 to 40 U mL-1 and a limit of detection of 0.07 U mL-1 using differential pulse voltammetry. In addition to detecting CA19-9 at clinically relevant concentrations for pancreatic cancer in standard solutions, the immunosensors provide the determination of CA19-9 on cell lysate and human serum samples. Using LbL films led to immunosensors with superior performance compared to similar systems obtained by drop casting. The fabrication of this relatively simple, inexpensive platform is a demonstration that SPCEs modified with cost-effective materials are able to detect cancer biomarkers and may be adapted to other disposable immunosensors. Graphical abstract Schematic representation of assembly and characterization of electrochemical immunosensors for the determination of carbohydrate antigen 19-9 based on printed electrodes modified with composites of carbon black and polyelectrolyte films.

Wearable potentiometric biosensor for analysis of urea in sweat.

Herein, we introduce wearable potentiometric biosensors on screen-printed carbon electrodes (SPCEs) for on-body and on-site monitoring of urea in sweat. The biosensor architecture was judiciously designed to detect urea at different pHs and incorporate a pH sensor, thus containing polyaniline ink, urease bioink and a polyvinylchloride membrane. Urea detection could be performed in the wide range from 5 to 200 mM at pH 7.0, encompassing urea levels in human sweat. The biosensor response was fast (incubation time 5 min), with no interference from other substances in sweat. Reliable urea detection could be done in undiluted human sweat with a skin-worn flexible device using the pH correction strategy afforded by the pH sensor. The performance of the epidermal biosensor was not affected by severe bending strains. The feasibility of mass production was demonstrated by fabricating epidermal flexible biosensors using slot-die coating with a roll-to-roll technique.

Fully-printed electrochemical sensors made with flexible screen-printed electrodes modified by roll-to-roll slot-die coating.

The manufacture of sensors using large-scale production techniques, such as roll-to-roll (R2R) processing, may fulfill requirements of low-cost disposable devices. Herein, we report the fabrication of fully-printed electrochemical sensors using screen-printed carbon electrodes coated with carbon black inks through slot-die coating within an R2R process. As a proof of concept, sensors were produced to detect the neurotransmitter dopamine with high reproducibility and low limit of detection (0.09 µmol L-1). Furthermore, fully-printed biosensors made with a tyrosinase-containing ink were used to detect catechol in natural water samples. Since slot-die deposition enables printing enzymes without significant activity loss, the biosensors exhibited high stability over a period of several weeks. Even more important, R2R slot-die coating may be extended to any type of sensors and biosensors with the possibility of large-scale manufacturing.

Electrical detection of pathogenic bacteria in food samples using information visualization methods with a sensor based on magnetic nanoparticles functionalized with antimicrobial peptides.

Outbreaks of foodborne diseases demand simple, rapid techniques for detecting pathogenic bacteria beyond the standard methods that are not applicable to routine analysis in the food industry and in the points of food consumption. In this work, we developed a sensitive, rapid and low-cost assay for detecting Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Salmonella typhimurium (S. typhi) in potable water and apple juice. The assay is based on electrical impedance spectroscopy measurements with screen-printed interdigitated electrodes coupled with magnetite nanoparticles functionalized with the antimicrobial peptide melittin (MLT). The data were analyzed with the information visualization methods Sammon's Mapping and Interactive Document Map to distinguish samples at two levels of contamination from food suitable for consumption. With this approach it has been possible to detect E. coli concentration down to 1 CFU mL-1 in potable water and 3.5 CFU mL-1 in apple juice without sample preparation, within only 25 min. This approach may serve as a low-cost, quick screening procedure to detect bacteria-related food poisoning, especially if the impedance data of several sensing units are combined.

Screen-printed interdigitated electrodes modified with nanostructured carbon nano-onion films for detecting the cancer biomarker CA19-9.

Nanostructured capacitive biosensors, combined with inexpensive fabrication technologies, may provide simple, sensitive devices for detecting clinically relevant cancer biomarkers. Herein, we report a novel platform for detecting the pancreatic cancer biomarker CA19-9 using low-cost screen-printed interdigitated electrodes (SPIDEs). The SPIDEs were modified by carbon nano-onions (CNOs) and graphene oxide (GO) films, on which a layer of anti-CA19-9 antibodies was immobilized. The modification with CNOs and GO significantly improved the analytical performance of the biosensor, which displayed superior results to those prepared only with GO. The biossensor exhibited high reproducibility and a relatively low limit of detection of 0.12 U mL-1. Using these devices in combination with information visualization methods we were able to detect CA19-9 in whole cell lysates of colorectal adenocarcinoma. The fabrication of these low-cost, disposable immunosensors is a successful attempt to explore CNOs in capacitive biosensors, which may be extended for detection of different cancer biomarkers.

Effect of carbon black functionalization on the analytical performance of a tyrosinase biosensor based on glassy carbon electrode modified with dihexadecylphosphate film.

Carbon Black (CB) has acquired a prominent position as a carbon nanomaterial for the development of electrochemical sensors and biosensors due to its low price and extraordinary electrochemical and physical properties. These properties are highly dependent on the surface chemistry and thus, the effect of functionalization has been widely studied for different applications. Meanwhile, the influence of CB functionalization over its properties for electroanalytical applications is still being poorly explored. In this study, we describe the use of chemically functionalized CB Vulcan XC 72R for the development of sensitive electrochemical biosensors. The chemical pre-treatment increased the material wettability by raising the concentration of surface oxygenated functional groups verified from elemental analysis and FTIR measurements. In addition, it was observed an enhancement of almost 100-fold on the electron transfer rate constant (k0) related to unfunctionalized CB, confirming a remarkable improvement of the electrocatalytic properties. Finally, we constructed a Tyrosinase (Tyr) biosensor based on functionalized CB and dihexadecylphosphate (DHP) for the determination of catechol in water samples. The resulting device displayed an excellent stability with a limit of detection of 8.7 × 10-8 mol L-1 and a sensitivity of 539 mA mol-1 L. Our results demonstrate that functionalized CB provides an excellent platform for biosensors development.