preparation-photoacoustic-analysis-cellular-vehicles-containing-gold

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Italian National Research Council

The DNA thrombin aptamer has been extensively investigated, and the coupling of this aptamer to different transduction principles has demonstrated the wide applicability of aptamers as bioreceptors in bioanalytical assays. The goal of this work was to design an aptamer-based sandwich assay with electrochemical detection for thrombin analysis in complex matrixes, using a simple target capturing step by aptamer-functionalized magnetic beads. The conditions for the aptamer immobilization and for the protein binding have been first optimized by surface plasmon resonance, and then transferred to the electrochemical-based assay performed onto screen-printed electrodes. The assay was then applied to the analysis of thrombin in buffer, spiked serum, and plasma and high sensitivity and specificity were found. Moreover, thrombin was generated in situ in plasma by the conversion of its precursor prothrombin, and the formation of thrombin was followed at different times. The concentrations detected by the electrochemical assay were in agreement with a simulation software that mimics the formation of thrombin over time (thrombogram). The proposed work demonstrates that the high specificity of aptamers together with the use of magnetic beads are the key features for aptamer-based analysis in complex matrixes, opening the possibility of a real application to diagnostics or medical investigation.

Aptamer-based detection of plasma proteins by an electrochemical assay coupled to magnetic beads.

Aptamer-based assays represent a modern and attractive approach in bioanalytical chemistry. The DNA thrombin aptamer has been extensively investigated, and the coupling of this aptamer to different transduction principles has demonstrated the wide applicability of aptamers as bioreceptors in bioanalytical assays. The goal of this work was to critically evaluate all the parameters that can influence the sensor performances by using the thrombin aptamer immobilized onto piezoelectric quartz crystals. The optimization of the immobilization and the binding protocol was of paramount importance, and improvements in analytical performances could be obtained by optimizing simple steps in immobilization and assay conditions. Moreover, the work demonstrated the possibility of using aptamer-based sensors in complex matrixes, opening the possibility of a real application to diagnostics or medical investigation.

Analytical performances of aptamer-based sensing for thrombin detection.

The main cause of reduced long-term graft survival is chronic allograft injury. Cardiovascular risk factors such as hyperhomocysteinaemia, accumulation of asymmetric dimethylarginine, increased oxidative stress and decreased production of nitric oxide seem to play an important role. Functional polymorphisms of the endothelial isoform of nitric oxide synthase (NOS) gene cause an alteration in nitric oxide production. Nitric oxide levels, and thus oxidative stress, are also influenced by hyperhomocysteinaemia.

The impact of eNOS, MTR and MTHFR polymorphisms on renal graft survival in children and young adults.

Results and perspectives on the biomedical exploitation of gold nanorods with plasmon resonances in the near infrared window are reported. The authors describe experimental studies of laser-activated nanoparticles in the direct welding of connective tissues, which may become a valuable technology in biomedicine. In particular, colloidal gold nanorods excited by diode laser radiation at 810 nm were used to mediate functional photothermal effects and weld eye's lens capsules and arteries. The preparation of biopolymeric matrices including gold nanorods is also described, as well as preliminary tests for their application in the closure of wounds in vessels and tendons. Finally, the use of these nanoparticles for future applications in the diagnosis, imaging and therapy of cancer is discussed.

Gold nanorods as new nanochromophores for photothermal therapies.

Chitosan films doped with gold nanorods as laser-activatable hybrid bioadhesives.

We evaluated the association between MCP-1, CCR2, RANTES, and CCR5 gene polymorphisms and upper urinary tract infection in 273 children recruited in Northeast Italy. Statistical analysis of RANTES-403 G>A genotype frequencies showed that children carrying the RANTES-403 G allele are at higher risk for urinary tract infection, irrespective of vesicoureteral reflux.

Upper urinary tract infections are associated with RANTES promoter polymorphism.

In this review, the current status of research in electrochemical immunosensors is considered. We primarily focus on label-free and enzyme-labeled immunosensors, and the analytical capabilities of these devices are discussed. Moreover, the use of magnetic beads as new materials for immunosensors coupled with electrochemical sensing is also described, together with the application of new molecules such as aptamers as specific biorecognition elements. Examples of the applicability of these devices in solving various analytical problems in clinical, environmental and food fields are reported. Finally, the prospects for the further development of immunosensor technologies are shown.

Strategies for electrochemical detection in immunochemistry.

In this paper we report on a new use for dark-field microscopy in order to retrieve two-dimensional maps of optical parameters of a thin sample such as a cryptograph, a histological section, or a cell monolayer. In particular, we discuss the construction of quantitative charts of light absorbance and scattering coefficients of a polyvinyl alcohol film that was embedded with gold nanorods and then etched using a focused mode-locked Ti:Sapphire oscillator. Individual pulses from this laser excite plasmonic oscillations of the gold nanorods, thus triggering plastic deformations of the particles and their environment, which are confined within a few hundred nm of the light focus. In turn, these deformations modify the light absorbance and scattering landscape, which can be measured with optical resolution in a dark-field microscope equipped with an objective of tuneable numerical aperture. This technique may prove to be valuable for various applications, such as the fast readout of optically encoded data or to model functional interactions between light and biological tissue at the level of cellular organelles, including the photothermolysis of cancer.

Quantitative readout of optically encoded gold nanorods using an ordinary dark-field microscope.

Mutations in human SIX1 gene cause branchiootorenal or branchiootic syndrome. Six1 deficient mice exhibit uni- or bilateral renal hypoplasia or kidney agenesis. Furthermore a lack of Six1 gene in the ureter leads to hydroureter and hydronephrosis. These murine malformations resemble human kidney and urinary tract congenital anomalies (CAKUT), a group of diseases with a diverse anatomical spectrum which includes duplex collecting system as much as urethra kidney and ureteropelvic anomalies. Our study focuses on whether mutations or deletion of this gene may be associated with nonsyndromic CAKUT.

SIX1 gene: absence of mutations in children with isolated congenital anomalies of kidney and urinary tract.

The NPHS2 gene encodes podocin, a membrane protein that acts as the structural scaffold in podocyte foot processes. NPHS2 mutations are associated with steroid-resistant nephrotic syndrome (SRNS), with the pathologic variant being focal and segmental glomerulosclerosis (FSGS), an emerging cause of end-stage renal disease in children. We describe a novel NPHS2 sequence variant in a girl with SRNS. Onset occurred at the age of seven years, with edema, hypo-proteinemia, hypoalbuminemia, hypercholesterolemia, hypertriglyceridemia and nephrotic proteinuria. Renal function was normal and autoimmunity markers were negative. Proteinuria failed to decrease after standard steroid therapy. Renal biopsy showed FSGS. Cyclosporine therapy was instituted, but no remission of proteinuria was achieved and chronic renal failure developed. Molecular analysis of the NPHS2 gene revealed a homozygous nucleotide substitution in position c.451+3A>T in intron 3-4. This nucleotide substitution has not been reported in the literature till date. The effect of the detected substitution on podocin protein was demonstrated by renal biopsy RNA extraction and cDNA amplification analysis. This technique had never been applied to a NPHS2 mutation. Based on these results, immunosuppressive drugs were discontinued and conservative therapy was undertaken.

mRNA sequencing of a novel NPHS2 intronic mutation in a child with focal and segmental glomerulosclerosis.

We present a darkfield optical microspectroscopy technique devoted to the disentangled measurement of the absorption and scattering cross sections of nanoparticle (NP) samples with variable concentration. The robustness of the method, including the needed instrumental calibrations, is examined in detail by analyzing and quantifying the major sources of statistic and systematic errors. As an exemplary case, results are presented on a gold NP colloid. The technique takes advantage of a simple inverted microscope, coupled with a spectrograph and equipped with a darkfield condenser and a variable numerical aperture objective to obtain spectra either in darkfield or brightfield optical configurations. By adopting the Lambert-Beer (LB) equation modeling, we were able to disentangle and measure with a single setup the absorption, scattering, and extinction coefficients of the same sample by combining three spectra, obtained by opportunely varying the objective numerical aperture. Typical plasmonic resonances were recognized at approximately 520 and 750 nm. Optical coefficients were measured as a function of particle number density (0.04-3.94 µm(-3), corresponding to 40 µM-4 mM nominal Au concentration) and good linearity was verified up to ∼1.5 µm(-3) (∼1 mM Au). Moreover, extinction and scattering cross sections were quantified and the validity of the LB approximation was reviewed. Besides its applications to plasmonic NPs, this method may be appropriate for any colloid, provided there exists a characteristic spectral feature in the ultraviolet-visible-near infrared range. This technique may be exploited to localize NPs in biological samples.

A simple method to disentangle nanoparticle optical properties by darkfield microspectroscopy.

The interest for gold nanorods in biomedical optics is driven by their intense absorbance of near infrared light, their biocompatibility and their potential to reach tumors after systemic administration. Examples of applications include the photoacoustic imaging and the photothermal ablation of cancer. In spite of great current efforts, the selective delivery of gold nanorods to tumors through the bloodstream remains a formidable challenge. Their bio-conjugation with targeting units, and in particular with antibodies, is perceived as a hopeful solution, but the complexity of living organisms complicates the identification of possible obstacles along the way to tumors.

In vitro assessment of antibody-conjugated gold nanorods for systemic injections.

SERS detection of proteins is typically performed by using labeling agents with stable and high Raman scattering cross sections. This is a valuable approach for trace detection and quantification of a target protein but is unsuitable for inspecting its inherent structural and functional properties. On the other hand, direct SERS of proteins has been mainly devoted to the study of short peptides and aminoacid sequences or of prosthetic groups with intense Raman signals, which is of scarce interest for a thorough characterization of most proteins. Here we try to overcome these limitations by setting-up an effective platform for the structural SERS analysis of proteins. The platform consists of an extended bidimensional array of gold concave nanocubes (CNCs) supported on a PDMS film. CNCs are closely-packed through face-face and face-corner interactions generating a monolayered arrangement featuring well distributed nanoholes. Here the protein homogeneously experiences an E-field enhancement outward from the metal surfaces surrounding it, which causes a large number of vibrations to be contemporarily amplified. The proposed platform provides stable and detailed SERS spectra and confers rapidity and reproducibility to the analysis.

Concave gold nanocube assemblies as nanotraps for surface-enhanced Raman scattering-based detection of proteins.

An electrochemical immunoassay for neopterin was developed using recently produced specific antibodies immobilized to protein A-coated magnetic beads in combination with differential pulse voltammetry and screen-printed array of electrodes. Neopterin-alkaline phosphatase conjugate was used as label in a competitive assay format. Multiplexed analysis of neopterin was demonstrated by replacing the traditional ELISA with electrochemical detection and the traditional plastic wells with screen-printed array of electrodes. The optimized electrochemical method, based on polyclonal antibodies, reached a limit of detection of 0.008 ng/mL with an average RSD %=10. Serum samples collected from patients with sepsis, healthy volunteers and other patients without a confirmed clinical diagnosis were also analyzed. The obtained results, compared with those of a commercial ELISA kit, had a significant correlation, showing the possibility to distinguish among the serum samples from ill or healthy subjects.

Detection of biomarkers for inflammatory diseases by an electrochemical immunoassay: the case of neopterin.

Gold nanorods (GNRs) are important platforms for biosensing and drug delivery. As for most nanomaterials, appropriate coatings such as polyethylene glycol (PEG) are needed to stabilize GNRs within biological fluids. We show here that the interactions of GNRs with proteins can be finely modulated through surface modification using PEG-containing chains bearing charged headgroups. Interestingly, introduction of amino or carboxylate groups produces relevant and differential changes in GNR interactions with three representative proteins: lysozyme, cytochrome c, and bovine serum albumin. These effects were explored through the direct monitoring of plasmonic bands of the GNRs and are supported by independent dynamic light scattering (DLS) and circular dichroism (CD) determinations. Notably, GNR-protein interactions observed for these charged GNRs can be almost completely reversed by salt addition. These observations demonstrate the importance of electrostatic effects in governing GNR-protein interactions, and provide a basis for new sensing and delivery platforms.

Article	Year
Aptamer-based detection of plasma proteins by an electrochemical assay coupled to magnetic beads. Analytical chemistry\| PubMed ID: 17297945	2007
Analytical performances of aptamer-based sensing for thrombin detection. Analytical chemistry\| PubMed ID: 17338547	2007
The impact of eNOS, MTR and MTHFR polymorphisms on renal graft survival in children and young adults. Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association\| PubMed ID: 19349296	2009
Gold nanorods as new nanochromophores for photothermal therapies. Journal of biophotonics\| PubMed ID: 20196029	2011
Chitosan films doped with gold nanorods as laser-activatable hybrid bioadhesives. Advanced materials (Deerfield Beach, Fla.)\| PubMed ID: 20734385	2010
Upper urinary tract infections are associated with RANTES promoter polymorphism. The Journal of pediatrics\| PubMed ID: 20869067	2010
Strategies for electrochemical detection in immunochemistry. Bioanalysis\| PubMed ID: 21083051	2009
Quantitative readout of optically encoded gold nanorods using an ordinary dark-field microscope. Nanoscale\| PubMed ID: 23832142	2013
SIX1 gene: absence of mutations in children with isolated congenital anomalies of kidney and urinary tract. Journal of nephrology\| PubMed ID: 24899122	2014
mRNA sequencing of a novel NPHS2 intronic mutation in a child with focal and segmental glomerulosclerosis. Saudi journal of kidney diseases and transplantation : an official publication of the Saudi Center for Organ Transplantation, Saudi Arabia\| PubMed ID: 24969201	2014
A simple method to disentangle nanoparticle optical properties by darkfield microspectroscopy. Microscopy research and technique\| PubMed ID: 25066896	2014
In vitro assessment of antibody-conjugated gold nanorods for systemic injections. Journal of nanobiotechnology\| PubMed ID: 25477237	2014
Concave gold nanocube assemblies as nanotraps for surface-enhanced Raman scattering-based detection of proteins. Nanoscale\| PubMed ID: 25563172	2015
Detection of biomarkers for inflammatory diseases by an electrochemical immunoassay: the case of neopterin. Talanta\| PubMed ID: 25618639	2015
Tuning the interactions of PEG-coated gold nanorods with BSA and model proteins through insertion of amino or carboxylate groups. Journal of inorganic biochemistry\| PubMed ID: 25978930	2015

Sonia Centi

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