Filippo Bosco

Surface-enhanced Raman spectroscopy based quantitative bioassay on aptamer-functionalized nanopillars using large-area Raman mapping.

Surface-enhanced Raman spectroscopy (SERS) has been used in a variety of biological applications due to its high sensitivity and specificity. Here, we report a SERS-based biosensing approach for quantitative detection of biomolecules. A SERS substrate bearing gold-decorated silicon nanopillars is functionalized with aptamers for sensitive and specific detection of target molecules. In this study, TAMRA-labeled vasopressin molecules in the picomolar regime (1 pM to 1 nM) are specifically captured by aptamers on the nanostructured SERS substrate and monitored by using an automated SERS signal mapping technique. From the experimental results, we show concentration-dependent SERS responses in the picomolar range by integrating SERS signal intensities over a scanning area. It is also noted that our signal mapping approach significantly improves statistical reproducibility and accounts for spot-to-spot variation in conventional SERS quantification. Furthermore, we have developed an analytical model capable of predicting experimental intensity distributions on the substrates for reliable quantification of biomolecules. Lastly, we have calculated the minimum needed area of Raman mapping for efficient and reliable analysis of each measurement. Combining our SERS mapping analysis with an aptamer-functionalized nanopillar substrate is found to be extremely efficient for detection of low-abundance biomolecules.

Quantification of rolling circle amplified DNA using magnetic nanobeads and a Blu-ray optical pick-up unit

We present the first implementation of a Blu-ray optical pickup unit (OPU) for the high-performance low-cost readout of a homogeneous assay in a multichamber microfluidic disc with a chamber thickness of 600 μm. The assay relies on optical measurements of the dynamics of magnetic nanobeads in an oscillating magnetic field applied along the light propagation direction. The laser light provided by the OPU is transmitted through the sample chamber and reflected back onto the photo detector array of the OPU via a mirror. Spectra of the 2nd harmonic photo detector signal vs. the frequency of the applied magnetic field show a characteristic peak due to freely rotating magnetic nanobeads. Beads bound to ~1 μm coils of DNA formed off-chip by padlock probe recognition and rolling circle amplification show a different dynamics and the intensity of the characteristic peak decreases. We have determined the optimum magnetic bead concentration to 0.1mg/mL and have measured the response vs. concentration of DNA coils formed from Escherichia Coli. We have found a limit of detection of 10 pM and a dynamic range of about two orders of magnitude, which is comparable to the performance obtained using costly and bulky laboratory equipment. The presented device leverages on the advanced but low-cost technology of Blu-ray OPUs to provide a low-cost and high-performance magnetic bead-based readout of homogeneous bioassays. The device is highly flexible and we have demonstrated its use on microfluidic chambers in a disc with a thickness compatible with current optical media mass-production facilities.

High throughput label-free platform for statistical bio-molecular sensing.

Sensors are crucial in many daily operations including security, environmental control, human diagnostics and patient monitoring. Screening and online monitoring require reliable and high-throughput sensing. We report on the demonstration of a high-throughput label-free sensor platform utilizing cantilever based sensors. These sensors have often been acclaimed to facilitate highly parallelized operation. Unfortunately, so far no concept has been presented which offers large datasets as well as easy liquid sample handling. We use optics and mechanics from a DVD player to handle liquid samples and to read-out cantilever deflection and resonant frequency. Also, surface roughness is measured. When combined with cantilever deflection the roughness is discovered to hold valuable additional information on specific and unspecific binding events. In a few minutes, 30 liquid samples can be analyzed in parallel, each by 24 cantilever-based sensors. The approach was used to detect the binding of streptavidin and antibodies.

Blu-ray based optomagnetic aptasensor for detection of small molecules

This paper describes an aptamer-based optomagnetic biosensor for detection of a small molecule based on target binding-induced inhibition of magnetic nanoparticle (MNP) clustering. For the detection of a target small molecule, two mutually exclusive binding reactions (aptamer-target binding and aptamer-DNA linker hybridization) are designed. An aptamer specific to the target and a DNA linker complementary to a part of the aptamer sequence are immobilized onto separate MNPs. Hybridization of the DNA linker and the aptamer induces formation of MNP clusters. The target-to-aptamer binding on MNPs prior to the addition of linker-functionalized MNPs significantly hinders the hybridization reaction, thus reducing the degree of MNP clustering. The clustering state, which is thus related to the target concentration, is then quantitatively determined by an optomagnetic readout technique that provides the hydrodynamic size distribution of MNPs and their clusters. A commercial Blu-ray optical pickup unit is used for optical signal acquisition, which enables the establishment of a low-cost and miniaturized biosensing platform. Experimental results show that the degree of MNP clustering correlates well with the concentration of a target small molecule, adenosine triphosphate (ATP) in this work, in the range between 10µM and 10mM. This successful proof-of-concept indicates that our optomagnetic aptasensor can be further developed as a low-cost biosensing platform for detection of small molecule biomarkers in an out-of-lab setting.

Integrating electrochemical detection with centrifugal microfluidics for real-time and fully automated sample testing

Here we present a robust, stable and low-noise experimental set-up for performing electrochemical detection on a centrifugal microfluidic platform. By using a low-noise electronic component (electrical slip-ring) it is possible to achieve continuous, on-line monitoring of electrochemical experiments, even when the microfluidic disc is spinning at high velocities. Automated sample handling is achieved by designing a microfluidic system to release analyte sequentially, utilizing on-disc passive valving. In addition, the microfluidic system is designed to trap and keep the liquid sample stationary during analysis. In this way it is possible to perform cyclic voltammetry (CV) measurements at varying spin speeds, without altering the electrochemical response. This greatly simplifies the interpretation and quantification of data. Finally, real-time and continuous monitoring of an entire electrochemical experiment, including all intermediate sample handling steps, is demonstrated by amperometric detection of on-disc mixing of analytes (PBS and ferricyanide).

Centrifugally driven microfluidic disc for detection of chromosomal translocations

Chromosome translocations are a common cause of congenital disorders and cancer. Current detection methods require use of expensive and highly specialized techniques to identify the chromosome regions involved in a translocation. There is a need for rapid yet specific detection for diagnosis and prognosis of patients. In this work we demonstrate a novel, centrifugally-driven microfluidic system for controlled manipulation of oligonucleotides and subsequent detection of chromosomal translocations. The device is fabricated in the form of a disc with capillary burst microvalves employed to control the fluid flow. The microvalves in series are designed to enable fluid movement from the center towards the periphery of the disc to handle DNA sequences representing translocation between chromosome 3 and 9. The translocation detection is performed in two hybridization steps in separate sorting and detection chambers. The burst frequencies of the two capillary burst microvalves are separated by 180 rpm enabling precise control of hybridization in each of the chambers. The DNA probes targeting a translocation are immobilized directly on PMMA by a UV-activated procedure, which is compatible with the disc fabrication method. The device performance was validated by successful specific hybridization of the translocation derivatives in the sorting and detection chambers.

Mathematical model for biomolecular quantification using large-area surface-enhanced Raman spectroscopy mapping

Surface-enhanced Raman spectroscopy (SERS) based on nanostructured platforms is a promising technique for quantitative and highly sensitive detection of biomolecules in the field of analytical biochemistry. Here, we report a mathematical model to predict experimental SERS signal (or hotspot) intensity distributions of target molecules on receptor-functionalized nanopillar substrates for biomolecular quantification. We demonstrate that by utilizing only a small set of empirically determined parameters, our general theoretical framework agrees with the experimental data particularly well in the picomolar concentration regimes. This developed model may be generally used for biomolecular quantification using Raman mapping on SERS substrates with planar geometries, in which the hotspots are approximated as electromagnetic enhancement fields generated by closely spaced dimers. Lastly, we also show that the detection limit of a specific target molecule, TAMRA-labeled vasopressin, approaches the single molecule level, thus opening up an exciting new chapter in the field of SERS quantification.

Nanomechanical recognition of prognostic biomarker suPAR with DVD-ROM optical technology.

In this work the use of a high-throughput nanomechanical detection system based on a DVD-ROM optical drive and cantilever sensors is presented for the detection of urokinase plasminogen activator receptor inflammatory biomarker (uPAR). Several large scale studies have linked elevated levels of soluble uPAR (suPAR) to infectious diseases, such as HIV, and certain types of cancer. Using hundreds of cantilevers and a DVD-based platform, cantilever deflection response from antibody-antigen recognition is investigated as a function of suPAR concentration. The goal is to provide a cheap and portable detection platform which can carry valuable prognostic information. In order to optimize the cantilever response the antibody immobilization and unspecific binding are initially characterized using quartz crystal microbalance technology. Also, the choice of antibody is explored in order to generate the largest surface stress on the cantilevers, thus increasing the signal. Using optimized experimental conditions the lowest detectable suPAR concentration is currently around 5 nM. The results reveal promising research strategies for the implementation of specific biochemical assays in a portable and high-throughput microsensor-based detection platform.

Xsense: a miniaturised multi-sensor platform for explosives detection

Realizing that no one sensing principle is perfect we set out to combine four fundamentally different sensing principles into one device. The reasoning is that each sensor will complement the others and provide redundancy under various environmental conditions. As each sensor can be fabricated using microfabrication the inherent advantages associated with MEMS technologies such as low fabrication costs and small device size allows us to integrate the four sensors into one portable device at a low cost.

An Astigmatic Detection System for Polymeric Cantilever-Based Sensors

We demonstrate the use of an astigmatic detection system (ADS) for resonance frequency identification of polymer microcantilever sensors. The ADS technology is based on a DVD optical head combined with an optical microscope (OM). The optical head has a signal bandwidth of 80 MHz, allowing thermal fluctuation measurements on cantilever beams with a subnanometer resolution. Furthermore, an external excitation can intensify the resonance amplitude, enhancing the signal- to-noise ratio. The full width at half maximum (FWHM) of the laser spot is 568 nm, which facilitates read-out on potentially submicrometer-sized cantilevers. The resonant frequency of SU-8 microcantilevers is measured by both thermal fluctuation and excited vibration measurement modes of the ADS.