Ultra-small dye-doped silica nanoparticles via modified sol-gel technique

In modern biosensing and imaging, fluorescence-based methods constitute the most diffused approach to achieve optimal detection of analytes, both in solution and on the single-particle level. Despite the huge progresses made in recent decades in the development of plasmonic biosensors and label-free sensing techniques, fluorescent molecules remain the most commonly used contrast agents to date for commercial imaging and detection methods. However, they exhibit low stability, can be difficult to functionalise, and often result in a low signal-to-noise ratio. Thus, embedding fluorescent probes into robust and bio-compatible materials, such as silica nanoparticles, can substantially enhance the detection limit and dramatically increase the sensitivity. In this work, ultra-small fluorescent silica nanoparticles (NPs) for optical biosensing applications were doped with a fluorescent dye, using simple water-based sol-gel approaches based on the classical Stober procedure. By systematically modulating reaction parameters, controllable size tuning of particle diameters as low as 10 nm was achieved. Particles morphology and optical response were evaluated showing a possible single-molecule behaviour, without employing microemulsion methods to achieve similar results

Aptamer based-microarraysfor protein detection: a potential tool for diagnostic applications

Aptamers are an emerging class of nucleic acid molecules with applications in several fields. They are RNA or single stranded DNA molecules selected for their folding in complex tridimensional structures: their conformation, encoded in their nucleotide sequence, determines the specific interaction with diverse target molecules in solution. Aptamers bind to specific protein with high specificity and affinity, making them attractive alternatives to the commonly used monoclonal antibodies both for therapeutic and diagnostic applications. Besides lower production costs, added advantages over antibodies are their relative ease of isolation and modifications, tailored binding affinity and reversible denaturation, making them suitable candidates for use in detection systems. We have developed a Sandwich Aptamer Microarray (SAM) for human thrombin detection immobilizing one aptamer on a glass slide (capture aptamer) and using a secondary aptamer for the detection of the analyte. This aptasensor represents a potential diagnostic system for thrombin and exploits two non-overlapping DNA antithrombin aptamers recognizing different exosites of the target protein, TBA1 and TBA2. The 15-mer aptamer TBA1 binds the fibrinogen binding site, whereas the 29-mer aptamer TBA2 binds the heparin binding domain of human thrombin. This distinct recognition pattern allows their use in tandem, since a ternary complex could possibly be formed by simultaneous recognition of thrombin. To adapt aptamers to a defined solid phase and to a specific detection technique, appropriate chemical modifications must be introduced. In the case of the microarray, immobilization to the solid support and labeling for detection imply precise chemistries that could sensibly alter the aptamer structure. An extensive analysis on the binary complexes formation has been therefore performed in solution by Electrophoretic Mobility Shift Assay (EMSA) to verify the sandwich complex formation by a Supershift assay. The system validated in solution was finally applied to the solid phase using an appropriate control and two different protocols for detection, resulting in an aptamer-based microarray for recognition of human thrombin with high specificity, using standard microarray slides and a fast and easy protocol. The aptamer-based biosensor could be used to analyze thrombin concentration in pathologic conditions, therefore representing a potential tool for diagnostic application

APTAMER MICROARRAY DEVELOPMENT FOR HUMAN THROMBIN DETECTION: INTERACTION ANALYSIS IN SOLUTION AND IN SOLID PHASE

Aptamers are RNA or single stranded DNA molecules that selectively bind to molecular targets with high specificity and affinity, making them attractive alternatives to the commonly used antibodies. Besides lower production costs, added advantages over antibodies are their relative ease of isolation and modifications, tailored binding affinity and reversible denaturation, making them suitable candidates for use as detection systems. An aptamer-based microarray for human thrombin detection has been developed, exploiting two non-overlapping DNA antithrombin aptamers recognizing different exosites of the target protein. TBA1 and TBA2 have been used: the 15-mer aptamer (TBA1) binds the fibrinogen binding site, whereas the 29-mer aptamer (TBA2) binds the heparin binding domain. This distinct recognition pattern allows their use in tandem, since a ternary complex could possibly be formed by simultaneous recognition of thrombin. When adapting aptamers to a defined solid phase and to a specific detection technique, appropriate and sometimes profound post-SELEX chemical modifications must be introduced. In the case of microarray, immobilization to the solid support and labeling for detection imply precise chemistries that could sensibly alter the aptamer structure. In fact, since aptamers are evolved in solution, any modification altering the chemical identity of aptamer could affect its folding and consequently its binding to the target: therefore, great care must be taken when developing aptamer-based detection methods in solid phase employing aptamers previously selected in solution. To investigate whether post-SELEX chemical modifications introduced in aptamers would affect thrombin recognition prior to the development of a Sandwich Aptamer Microarray (SAM) system, we adopted these two aptamers and human thrombin as a model system: an extensive analysis on the binary complexes formation has been performed in solution by Electrophoresis Mobility Shift Assay (EMSA) and the verification of the sandwich complex formation in solution by a Supershift assay, incubating simultaneously the two aptamers with the target protein. The validated system was finally applied to the solid phase using an appropriate control and two different protocols for detection. An aptamer-based microarray for thrombin detection has therefore been created, in a sandwich-type format, for recognition of human thrombin with high specificity, using standard microarray slides and a fast and easy protocol. The aptamer-based biosensor could be used to analyze thrombin plasmatic concentration in pathologic conditions, using an easy and reliable protocol

Sewage monitoring for pathogenic microrganisms detection by using a low density microarray

Aims. Sewages are highly contaminated by numerous biological agents: bacteria, protozoa, fungi and viruses. Some of them can be very resistant to treatments and remain at high concentration in the effluent, thus representing an hazard especially in the case of water reuse for agriculture. Molecular methods are almost an important way to detect these pathogens, in particular the most applied techniques are based on protocols of nucleic acid amplification and quantification, of which the quantitative polymerase chain reaction (qPCR) is rapidly becoming established in the environmental sector for its higher sensitivity in comparison with cultural assays. Moreover the possibility in using molecular methods for simultaneous and rapid multiple detection of pathogens could be useful for risk assessment or safety purposes. This approach could be now possible using the technological advances of the DNA microarray, born for clinical analysis and based on the simultaneous qualitative analysis for multiple genera, species and strains. The large number of DNA sequences that can be spotted on a microarray together with the high specificity of binding to the immobilised sequence targets, allows the detection of a large range of microorganisms with high discriminatory ability. Nevertheless the application of this technology requires a deep study of its sensitivity and specificity. In this work, an environmental monitoring of sewage, sampled from a Waste Water Treatment Plant (WWTP), was performed in order to detect some different viruses and bacteria frequently present: Human Adenovirus (HAdV), Norovirus GGII (NV), Hepatitis A Virus (HAV), Enterovirus (EV), Rotavirus (RV), Enterohemorrhagic E. coli (EHEC), Salmonella enterica (SE) using a low density microarray containing specific oligonucleotides sequences for each of them. Methods. In the first phase of the study, a series of trials were performed to choose the sample treatment that was able to increase the sensitivity of molecular method. In particular 10 sewage samples (10 L) were treated with a first step based on tangential ultrafiltration followed by a second step in which the obtained eluates were further concentrated by ultracentrifugation until a final volume of 1 ml. After each step, the obtained eluates were treated with commercial kit (QIAgen) to extract viral and bacteria nucleic acids (NA) that were quantified by separate and specific qPCR reactions (Genomic Copies - GC) according to published protocols. In the second phase, an environmental monitoring was performed for 1 year from June 2013 to June 2014 by monthly sampling of 10 L of sewage from a WWTP in Northern Italy (13 samples). The concentrated samples, obtained by the previous phase chosen method, were treated to extract NA that were subsequently concentrated by speed VAC, labelled and overnight hybridised on the microarray slides. After that, a scanner read the positive signal. Parallel, aliquots of extracted NA of each samples were quantify (GC/10 L) by qPCR reactions. Results. The data of the trials revealed that the combination of ultrafiltration-ultracentrifugation permitted to increase the mean concentration of target microorganisms of 1-2 Log in comparison with the use of only ultrafiltration method. The microarray analysis revealed the presence of HAdv, EV, EHEC and SE in tested samples, while no NV, HAV and RV were detected. In particular, 78% (10/13) of samples resulted positive for HAdV, 30% (4/13) for EV, 84% (11/13) for EHEH and 61% (8/13) for SE. The simultaneous presence in the same sample of all these 4 target was detected in 2 samples (15%), of three target in 6 samples (46%) and of two target in 2 of samples (15%). The mean concentration of positive target estimated by qPCR were 2 x 108 GC/10 L for HAdV, 4 x 108 CG/10 L for EV, 107 GC/10 L for EHEC and 3 x 105 GC/10 L for SE. These data are in according of sewage microbial concentration published by several authors (Figure 1). The absence of positive samples for NV, HAV and RV was probably due by epidemiological situation in the monitored region. The data, moreover, underlined a limit of sensitivity of the test: samples with a target concentration lower than 2,8 x 103 GC/10 L resulted negative to microarray. Conclusion. The results of environmental monitoring were very promising for a multiple detection of pathogens in sewage confirming the possible use of microarray as a tool for screening. The major limitation of this technique was the scarce sensitivity that can be improved with specific sample treatments permitting also the purification of samples, as made in this study, by the combination of ultrafiltration and ultracentrifugation

SPECTRA: A Novel Compact System for Surface Plasmon Resonance Measurements

Surface plasmon resonance (SPR) is a common and useful measurement technique to perform fast and sensitive optical detection. SPR instrumentations usually comprise optical systems of mirrors and lenses which are quite expensive and impractical for point-of-care applications. In this work, we presented a novel and compact SPR device called SPECTRA, designed as a spectrophotometer add-on with a grating coupling configuration. The device is conceived as a marketable solution to perform quick SPR measurements in grating configuration without the requirement of complex instrumentation. The device can be customized either in a vertical structure to reach lower incident light angles, or in a horizontal configuration, which is suitable for SPR analysis using liquid solutions. The SPECTRA performance was evaluated through SPR measurements in typical applications. The vertical SPECTRA system was employed to detect different functionalization molecules on gold 720 nm-period grating devices. Meanwhile, the horizontal SPECTRA configuration was exploited to carry out fluid-dynamic measurements using a microfluidic cell with glycerol solutions at increasing concentrations to account for different refractive indexes. The experimental tests confirmed that the SPECTRA design is suitable for SPR measurements, demonstrating its capability to detect the presence of analytes and changes in surface properties both in static and dynamic set-ups

An Electrochemical Biosensor for the Detection of Bacteriophage of Lactococcus Lactis

The bacterial activity of Lactococcus lactis is exploited in the dairy industry for the fermentation of milk. However, the spreading of phages of L. lactis prevents the proper lactic fermentation, causing economic losses and waste. This work aims to explore an electrochemical detection method for the fast and reliable monitoring of the presence of the L. lactis phages. The detection is based on electrochemical biosensor with live L. lactis bacteria covering the sensor electrodes, whose electrical response is measured by electrochemical impedance spectroscopy (EIS). Solutions contaminated by phages induce bacteria lysis, clearly reducing the bacteria coverage over the electrodes and leading to evident parametrical shifts in the charge transfer resistance. Screen-printed and interdigitated sensors were tested to identify the most stable electrode suitable to work with the lab solutions. Experimental measurements with laboratory contaminated ideal samples show that the screen-printed sensors with gold and silver electrodes have great stability, reproducibility, and detection capability. Electrochemical results evidence great decreases of the charge transfer resistance in phages contaminated sensors in lab environments, paving the basis for the development of a new electrochemical biosensor capable of detecting the L. lactis phages

Enhancing the Sensitivity of DNA Microarray Using Dye-Doped Silica Nanoparticles: Detection of Human Papilloma Virus

DNA microarray is a high-throughput technology used for detection and quantification of nucleic acid molecules and others of biological interest. The analysis is based on the specific hybridization between probe sequences deposited in array and a target ss-DNA amplified by PCR and functionalized by a fluorescent dye. Organic labels have well known disadvantages like photobleaching and low signal intensities, which put a limitation to the lower amount of DNA material that can be detected. Therefore for trace analysis the development of more efficient biomarkers is required. With this aim we present in this paper the synthesis and application of alternative hybrid nanosystems obtained by incorporating standard fluorescent molecules into monodisperse silica nanoparticles. Efficient application to the detection of Human Papilloma Virus is demonstrated. This virus is associated to the formation of cervical cancer, a leading cause of death by cancer for women worldwide. It is shown that the use of the novel biomarkers increases the optical signal of about one order of magnitude with respect to the free dyes or quantum dots in conventional instruments. This is due to the high number of molecules that can be accommodated into each nanoparticle, to the reduced photobleaching and to the improved environmental protection of the dyes when encapsulated in the silica matrix. The cheap and easy synthesis of these luminescent particles, the stability in water, the surface functionalizability and bio-compatibility make them very promising for present and future bio-labeling and bio-imaging applications