Thiol-click photochemistry for surface functionalization applied to optical biosensing.

In the field of biosensing, suitable procedures for efficient probes immobilization are of outmost importance. Here we present different light-based strategies to promote the covalent attachment of thiolated capture probes (oligonucleotides and proteins) on different materials and working formats. One strategy employs epoxylated surfaces and uses the light to accomplish the ring opening by a thiol moiety present in a probe. However, most of this work lies on the use of thiol-ene photocoupling chemistry to covalently attach probes to the supports. And thus, both alkenyl and thiol derivatized surfaces are assayed to immobilize thiol or alkene ended probes, respectively, and their performances are compared. Also, the effect of the number of thiols carried by the probe is analyzed comparing single-point and multi-point attachment. The performance of the analogous tethering, but onto alkynylated surfaces is also carried out, and the sensing response is related to the surfaces hydrophobicity. A newly developed reaction is also discussed where a fluorinated surface undergoes the covalent immobilization of thiolated probes activated by light, creating small hydrophilic areas where the probes are attached, and leaving the rest of the surface highly hydrophobic and repellent against protein unspecific adsorption. These mixed surfaces confine the sample (aqueous) uniquely on the hydrophilic spots lowering the background signal and thus increasing the sensitivity. These probe immobilization approaches are applied to fluorescence microarray and label-free nanophotonic biosensing. All the exposed reactions have in common the photoactivation of the thiol moieties, and give rise to quick, clean, versatile, orthogonal and biocompatible reactions. Water is the only solvent used, and light the only catalyzer applied. Thus, all of them can be considered as having the attributes of click-chemistry reactions. For these reasons we named them as thiol-click photochemistry, being a very interesting pool of possibilities when building a biosensor.

Modulating receptor-ligand binding in biorecognition by setting surface wettability.

Modulation of support wettability used for microarray format biosensing has led to an improvement of results. Hydrophobicity of glass chips was set by derivatizing with single vinyl organosilanes of different chain length and silane mixtures. Thiol-ene photochemical linking has been used as effective chemistry for covalent anchoring of thiolated probes. Lowest unspecific binding and highest signal intensity and SNR were obtained with large hydrocarbon chain (C22) silanes or a shorter one (C10) containing fluorine atoms. SNR resulting values are improved, reaching levels higher than 1500 in some cases, when using vinyl silanes modified with 1% C10 alkyl fluorinated one, because mild hydrophobicity was achieved (water contact angle ca. 110°) for all silanes, including the short C2 and C3, thus giving rise to smaller and better defined array spots. In addition, unspecific binding of reagents and targets was totally withdrawn. Hence, good-performing surfaces for biosensing applications can be built using appropriate organosilane reagent selection, including fluorinated ones. Graphical abstract ᅟ.

High density microarrays on Blu-ray discs for massive screening.

The potential and the capabilities of Blu-ray technology (discs and drives) for massive screening applications are presented. High density microarrays are fabricated onto a Blu-ray disc and applied for the determination of microcystin residues and pathogenic microorganisms. Specific probes were physisorbed onto the BD surface and the biorecognition event was displayed using labeled secondary antibody solution and subsequent signal amplification. The attenuation of the reflected light caused by the reaction product is detected by the Blu-ray drive and inversely correlated with analyte concentration. BDs preserve the optical properties according to Blu-ray specifications, ensuring maximum accuracy and sensitivity of the drive during disc scanning. Detection limits of 0.4 µg/L for microcystin LR and 10(0) and 10(1) cfu/mL for Salmonella typhimurium and Cronobacter sakazakii respectively, were achieved, improving considerably the DVD performances and reaching similar sensitivity as real-time quantitative PCR. Blu-ray technology adapted to the analysis of high density arrays highlights the enormous capabilities (high sensitivity, speed-scanning, optical resolution, portability) for point-of-care settings, diagnostics, and high-throughput screening applications.

INSEL: an in silico method for optimizing and exploring biorecognition assays.

A practical in silico method for optimizing and exploring biointeraction-based events is developed.

Advanced homogeneous-heterogeneous immunosensing format employing restricted access supports.

A rapid immunosensing methodology that employs the so-called homogeneous-heterogeneous assay mode is presented. The immunosensor is based on the homogeneous competition among the analyte, a fluorescent tracer, and the antibody, followed by separation of free and bound species by means of a restricted access alkyl-diol silica C18 reversed-phase chromatographic support. In order to develop a general labeling methodology, fluorescent tracers are synthesized from oligonucleotides covalently bound to the hapten in 3' position and the marker in 5'. The immunosensor principle is demonstrated by determining atrazine in a completely automated manner at 2 min/sample without regeneration of the support and a limit of detection of 1.0 microg/L with the optimized system. Preliminary assays employing multilabeled tracers indicate that sensitivity can be improved. Organic solvents 2-propanol and acetonitrile up to 15% (v/v) are well tolerated, while methanol can be added to 50%. The sensor capabilities are demonstrated through the analysis of natural waters.

Glyphosate immunosensor. Application for water and soil analysis.

A fully automated immunosensor for the herbicide glyphosate has been developed on the basis of the immunocomplex capture assay protocol. The sensor carries out on-line analyte derivatization prior to the assay and uses a selective anti-glyphosate serum, a glyphosate peroxidase enzyme tracer, and fluorescent detection. Under optimal conditions, the detection limit achieved is 0.021 microg/L with an analysis rate of 25 min per assay, autonomy of more than 48 h, and sensor reusability >500 analytical cycles. The immunosensor is able to discriminate structurally related molecules, such as aminomethylphosphonic acid, the main metabolite of glyphosate, and other related herbicides, such as glufosinate and glyphosine. Interferences from naturally occurring species (anions, cations, and humic substances) and their elimination were also studied. The immunosensor has been successfully applied to water and soil sample analysis, with good recoveries at levels lower than 1 microg/L. Results obtained with the immunosensor correlate well with data from a magnetic particle ELISA and LC/LC/MS chromatographic method.

Rapid immunoanalytical method for the determination of atrazine residues in olive oil.

A sensitive, simple and reliable method has been developed for the determination of atrazine in extra virgin olive oil. The analytical procedure involves direct extraction of the target analyte from oil matrix with methanol and a freezing clean-up step (-80 degrees C) followed by plate or sensor immunoassay determination. A detection limit of 0.7 ng/mL, with a dynamic range from 1.0 to 10.4 ng/mL, was reached. The method was highly selective for atrazine and propazine, showing little or no cross-reactivity to other similar compounds. The excellent recoveries obtained (mean value 91.3%) confirm the potential of this approach to detect atrazine in olive oil for application as screening and complementary method in pesticide regulatory and food safety programs. The proposed method correlates well with the reference gas chromatography (GC-MS) technique.