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.

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.

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.

Quantitative detection of human tumor necrosis factor α by a resonance raman enzyme-linked immunosorbent assay.

Tumor necrosis factor α is an inflammatory cytokine which has been linked with many infectious and inflammatory diseases. Detection and quantification of this key biomarker is commonly achieved by use of an enzyme-linked immunosorbent assay (ELISA). This fundamental technique uses the spectroscopic detection of a chromogen such as 3,3',5,5'-tetramethylbenzidine (TMB). Horseradish peroxidase (HRP), bound to the detection antibody, catalyzes the oxidation of TMB by hydrogen peroxide to generate colored products which may be measured spectrophotometrically. In this study we have used a conventional ELISA kit and shown that, by replacing the traditional colorimetric detection with resonance Raman spectroscopy, we can achieve 50 times lower detection limits and the potential for multiplexed analysis is increased. In this approach, the laser wavelength was tuned to be in resonance with an electronic transition of the oxidized TMB. The relative intensity of the enhanced Raman bands is proportional to the amount of TMB, thus providing a means of improved quantification. Furthermore, TMB is one of the most widely used chromogenic substrates for HRP-based detection and commercial ELISA test kits, indicating that this detection technique is applicable to a large number of target analytes.