Aaron Hernandez-Santana

SERRS labelled beads for multiplex detection.

Beads labelled using surface enhanced resonance Raman scattering (SERRS) are highly sensitive and specific tags, with potential applications in biological assays, including molecular diagnostics. The beads consist of a nucleus containing dye labelled silver-nanoparticle aggregates surrounded by a polymer core. The nuclei generate strong SERRS signals. To illustrate the coding advantage created by the sharp, molecularly specific SERRS signals, four specially designed SERRS dyes have been used as labels and three of these have been combined in a multiplex analysis. These dyes use specific groups such as benzotriazole and 8-hydroxyquinoline to improve binding to the surface of the silver particles. The aggregation state of the particles is held constant by the polymer core, this nucleus also contains many dye labels, yielding a very high Raman scattering intensity for each bead. To functionalise these beads for use in biological assays an outer polymer shell can be added, which allows the attachment of oligonucleotide probes. Oligonucleotide modified beads can then be used for detection of specific oligonucleotide targets. The specificity of SERRS will allow for the detection of multiple targets within a single assay.

Rapid prototyping of poly(dimethoxysiloxane) dot arrays by dip-pen nanolithography

We report the first direct patterning of elastomeric PDMS structures by dip-pen nanolithography (DPN). This method involves the use of a cantilever tip to transfer a PDMS ink onto a silicon dioxide surface to create dot array patterns which are then cross-linked and bonded irreversibly to the substrate. The chemical composition of the PDMS structures deposited by DPN was characterised by Raman microspectroscopy to provide an insight into the ink transfer process. This technique offers a significant advance in the ability to rapidly and easily produce programmable surface features from a widely used polymer for use in a variety of applications.

Fabricating protein immunoassay arrays on nitrocellulose using Dip-pen lithography techniques

Advancements in lithography methods for printing biomolecules on surfaces are proving to be potentially beneficial for disease screening and biological research. Dip-pen nanolithography (DPN) is a versatile micro and nanofabrication technique that has the ability to produce functional biomolecule arrays. The greatest advantage, with respect to the printing mechanism, is that DPN adheres to the sensitive mild conditions required for biomolecules such as proteins. We have developed an optimised, high-throughput printing technique for fabricating protein arrays using DPN. This study highlights the fabrication of a prostate specific antigen (PSA) immunoassay detectable by fluorescence. Spot sizes are typically no larger than 8 μm in diameter and limits of detection for PSA are comparable with a commercially available ELISA kit. Furthermore, atomic force microscopy (AFM) analysis of the array surface gives great insight into how the nitrocellulose substrate functions to retain protein integrity. This is the first report of protein arrays being printed on nitrocellulose using the DPN technique and the smallest feature size yet to be achieved on this type of surface. This method offers a significant advance in the ability to produce dense protein arrays on nitrocellulose which are suitable for disease screening using standard fluorescence detection.

Tracking Bisphosphonates through a 20 mm Thick Porcine Tissue by Using Surface-Enhanced Spatially Offset Raman Spectroscopy†

Track it down: A recognized surface-enhanced Raman scattering (SERS) nanotag signal was monitored from a thin, dispersed layer of bisphosphonate-functionalized nanotags on a bone sample, through a 20 mm thick specimen of porcine muscle tissue by surface-enhanced spatial offset Raman spectroscopy (SESORS; see picture). The result demonstrates the great potential for non-invasive in vivo bisphosphonate drug tracking.

Immunoassay arrays fabricated by dip-pen nanolithography with resonance Raman detection.

Here, we report the first use of resonance Raman scattering for the detection of miniaturized microscale arrays fabricated by dip-pen nanolithography. Antibody arrays for prostate-specific antigen (PSA) were printed, and a sandwich immunoassay was carried out. An enzyme-linked detection antibody was used to provide an insoluble and stable colored microdot in the recommended size range for microarray readers, which could be read with resonance Raman scattering. This gives quantitative detection as well as an improved detection limit and a larger dynamic range than that previously achieved by direct fluorescent detection methods. By Raman mapping across the arrayed area, the microdots were easily detected with very little background signal from surrounding areas. Levels of PSA as low as 25 pg/mL were detected using this method, which could be extended to a large number of useful biomarkers.

Determination of calcium in synovial fluid samples as an aid to diagnosing osteoarthritis

BACKGROUND Microscopic inorganic crystals are commonly observed in the synovial fluid of patients suffering from arthritic diseases. Basic calcium phosphate (BCP) crystals are known to occur quite commonly in the joint fluid of osteoarthritis (OA) patients and are insoluble at physiological pH. Current analysis of patient synovial fluid depends on light microscopy and staining with Alizarin Red-S. Both methods cannot identify crystals < 1µm in size and are highly subjective. This article investigates the use of o-cresolphthalein complexone (OCP), a colorimetric reagent, to quantify calcium from crystals isolated from synovial fluid samples as a means of identifying the presence of BCP and, hence, improving the diagnosis of OA. RESULTS Inorganic crystals were isolated following degradation of the biological sample matrix with hyaluronidase. 1-M HNO(3) was used for crystal dissociation into ions and the colorimetric response of OCP to calcium was measured in a basic environment of 2-amino-2-methyl-1-propanol. The average calcium content in OA patient samples was up to 40% higher than in rheumatoid arthritis (RA) patient samples. RA samples were used as a comparison, because they are generally accepted to be crystal free. Within the OA group, higher levels of calcium were detected in three out of 12 synovial fluid samples, which correlated with a significantly greater number of BCP crystals detected during microscopic examination. CONCLUSIONS A simple method based on colorimetry for measurement of calcium content and semiquantification of BCP crystals in synovial fluid samples has been described. Sample pretreatment following addition of hyaluronidase proved to be effective in reducing viscosity and aiding the dissociation of BCP crystals in synovial fluid samples.

The inorganic chemistry of surface enhanced raman scattering (SERS)

Surface enhanced Raman scattering (SERS) has enormous potential as a detection technique. Used correctly, it can combine many of the advantages of Raman spectroscopy such as stand off detection and selective identification of a species in situ, with the additional advantages of high sensitivity and ...

Multidentate macromolecules for functionalisation, passivation and labelling of metal nanoparticles

A new class of SERRS-active macromolecule designed to protect silver nanoparticle surfaces against salt corrosion whilst retaining colloidal stability of the particles is reported.

Dip-pen nanolithography of nanostructured oligofluorene truxenes in a photo-curable host matrix

We report the controlled patterning of nano-sized oligofluorene truxenes onto silicon dioxide by dip-pen nanolithography (DPN) using a UV-curable pre-polymer as a carrier fluid. In this technique, a sharp atomic force microscope (AFM) cantilever tip is used to transfer the liquid ink onto a surface using piezo-controlled movements and excellent spatial registry. The photo-curable carrier fluid is then exposed to UV-light to produce a cross-linked, host matrix while retaining the photoluminescent properties of the truxenes and providing protection against photo-oxidation. The chemical composition of the composite structures deposited by DPN was characterised by Raman microspectroscopy and microphotoluminescence to demonstrate successful incorporation of the photoluminescent truxenes in the polymer matrix.

Young Investigator: Aaron Hernandez-Santana

Supervisor’s supporting comments Dr Aaron Hernandez-Santana is a creative, dynamic and enthusiastic researcher and I know that he will go far in his career, wherever that may take him. His work in the Bioanalytical Chemistry and Diagnostics research group at Dublin City University, Ireland, has been innovative and he uses his experience in new and exciting ways, meaning he always brings value to his research. Aaron is truly a problem-solving bioanalytical investigator – he works easily with other disciplines, he applies his chemistry knowledge to physics and biology applications, he is not afraid to move outside his comfort zone of research expertise, acquiring new skills as and when he needs them and he has some great ideas that will serve him well in the future.