comparative-analysis-human-growth-hormone-serum-using-spri-nano-spri

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

HORIBA Scientific

Metallothionein fusion proteins allow for site-specific, orthogonal functionalization of proteins to a variety of nanoparticles.

General, high-affinity approach for the synthesis of fluorophore appended protein nanoparticle assemblies.

Metalloprotein tethered CdSe nanoparticles have been generated to provide selective and reagentless maltose biosensing. As opposed to cell or protein detection by semiconducting nanoparticle bioconjugates, a modular method for small-molecule detection using semiconducting nanoparticle bioconjugates has been difficult. Here we report a method for reagentless protein-based semiconducting nanoparticle biosensors. This method uses Ru(II) complex-CdSe nanoparticle interactions and the maltose-induced conformation changes of maltose binding protein to alter the CdSe nanoparticle fluorescence emission intensity. In this proof-of-principle system, the maltose-induced protein conformation changes alter the Ru(II) complex-CdSe nanoparticle interaction, which increases the CdSe emission intensity. Altered CdSe emission intensity effects are best described as electron transfer from the Ru(II) complex to the CdSe excited state forming the nonfluorescent CdSe anion. Four surface-cysteine, Ru(II) complex-attached maltose-binding proteins have been studied for maltose dependent alteration of CdSe emission intensities. With 3.0-3.5 nm diameter CdSe nanoparticles, all ruthenated maltose-binding proteins display similar maltose-dependent increases (1.4-fold) in CdSe emission intensity and maltose binding affinities (KA = 3 x 106 M-1). For these four systems, the only difference was the sample-to-sample variation in maltose-dependent responses. Thus, very few surface cysteine mutations need to be examined to find a successful biosensor, as opposed to analogous systems using organic fluorophores. This strategy generates a unimolecular, or reagentless, semiconducting nanoparticle biosensor for maltose, which could be applied to other proteins with ligand-dependent conformation changes.

A modular nanoparticle-based system for reagentless small molecule biosensing.

Reagentless and reversible maltose biosensors are demonstrated using ZnS coated CdSe (CdSe@ZnS) nanoparticle emission intensities. This method is based on electron transfer quenching of unimolecular protein-CdSe@ZnS nanoparticle assemblies, which is provided by a protein-attached Ru(II) complex. This Ru(II) complex is presumed to reduce a valence band hole of the CdSe@ZnS excited state by tunneling through the ZnS overcoating. The Ru(II) complex mediated quenching of CdSe@ZnS nanoparticle emission was only decreased 1.2-fold relative to the CdSe nanoparticle systems. While four different Ru(II) complex attachment sites provided different amounts of nanoparticle emission quenching (1.20 to 1.75-fold decrease), all of these attachment sites yielded maltose-dependent intensity changes (1.1 to 1.4-fold increase upon maltose addition). Maltose dissociation constants for these four biosensing systems range from 250 nM to 1.0 microM, which are similar to the maltose-maltose binding protein dissociation constant that these sensors are based on. The increased fluorescence intensity was found to only occur in the presence of maltose. Furthermore, the ability of these reagentless protein-nanoparticle assemblies to perform maltose biosensing reversibly is demonstrated with the addition of alpha-glucosidase. Three 50 microM maltose additions after alpha-glucosidase addition showed increases of 2.2 microM, 600 nM, and 150 nM maltose. This result demonstrates a fluorometric method for examining alpha-glucosidase activity. Using maltose binding protein to control Ru(II) complex interactions with CdSe@ZnS nanoparticle surfaces provide a novel class of highly fluorescent, photostable biosensors that are selective for maltose.

Selective, reversible, reagentless maltose biosensing with core-shell semiconducting nanoparticles.

Metallothioneins initiate semiconducting nanoparticle cellular toxicity.

Prodrugs are biologically inactive derivatives of an active drug intended to solve certain problems of the parent drug such as toxicity, instability, minimal solubility and non-targeting capabilities. The majority of drugs for cardiovascular diseases undergo first-pass metabolism, resulting in drug inactivation and generation of toxic metabolites, which makes them appealing targets for prodrug design. Since prodrugs undergo a chemical reaction to form the parent drug once inside the body, this makes them very effective in controlling the release of a variety of compounds to the targeted site. This review will provide the reader with an insight on the latest developments of prodrugs that are available for treating a variety of cardiovascular diseases. In addition, we will focus on several drug delivery methodologies that have merged with the prodrug approach to provide enhanced target specificity and controlled drug release with minimal side effects.

Prodrugs in cardiovascular therapy.

To assess the effects of oleic acid treatment on subcellular distribution of indium gallium phosphide-zinc sulfide (InGaP/ZnS) nanoparticles in microglia and astrocytes.

Imaging and organelle distribution of fluorescent InGaP/ZnS nanoparticles in glial cells.

The surface plasmon resonance imaging chip biointerface is fully designed using near-infrared (NIR) quantum dots (QDs) for the enhancement of surface plasmon resonance imaging (SPRi) signals in order to extend their application for medical diagnostics. The measured SPRi detection signal following the QD binding to the surface was amplified 25-fold for a 1 nM concentration of single-stranded DNA (ssDNA) and 50-fold for a 1 μg/mL concentration of prostate-specific antigen (PSA), a cancer biomarker, thus substantiating their wide potential to study interactions of a diverse set of small biomolecules. This significant enhancement is attributed to the QD's mass-loading effect and spontaneous emission coupling with propagating surface plasmons, which allowed the SPRi limit of detection to be reduced to 100 fM and 100 pg/mL for ssDNA and PSA, respectively. Furthermore, this study illustrates the potential of SPRi to be easily integrated with fluorescent imaging for advanced correlative surface-interaction analysis.

Designed biointerface using near-infrared quantum dots for ultrasensitive surface plasmon resonance imaging biosensors.

To enhance the stability of siRNA while improving their therapeutic properties and visualization at the target site, a novel nanoplex system was developed.

Self-assembled multifunctional nanoplexes for gene inhibitory therapy.

The hallmark of atherosclerosis is the accumulation of plaque in vessel walls. This process is initiated when monocytic cells differentiate into macrophage foam cells under conditions with high levels of atherogenic lipoproteins. Vulnerable plaque can dislodge, enter the blood stream, and result in acute myocardial infarction and stroke. Imaging techniques such as cardiovascular magnetic resonance (CMR) provides one strategy to identify patients with plaque accumulation.

Functionalization of gadolinium metallofullerenes for detecting atherosclerotic plaque lesions by cardiovascular magnetic resonance.

Diagnostic biomarkers (i.e. proteins) are often in low abundance in bodily fluids presenting many challenges for their detection. In order to extend the application of SPRi systems in detecting biomarkers at ultralow levels, we combine the advantage of aptamer technology with nanomaterials and microwave-assisted surface functionalization. By implementing a sandwich assay through the introduction of aptamer-modified quantum dots (QDs), it was possible to measure 7 zeptomole (at 5 fg/mL) of C-reactive protein (CRP) selectively in spiked human serum. It is expected that the proposed platform will provide new direction in designing ultrasensitive SPRi biosensors with multiplexing capabilities.

Zeptomole detection of C-reactive protein in serum by a nanoparticle amplified surface plasmon resonance imaging aptasensor.

The fabrication of 4-mercaptobenzoic acid (4-MBA) antibody-functionalized gold nanoparticles via microwave technology for surface-enhanced Raman scattering (SERS)-based cellular nanosensing is reported. Nanoprobes were characterized by UV-vis absorbance, Raman scattering properties, and observed by TEM imaging. Results showed that microwave irradiation rapidly yielded nanoprobes with significant Raman scattering intensity and suitable stability to support antibody conjugation in under 10min. Functionalized nanoprobes demonstrated the ability to map the expression of vascular adhesion molecule-1 (VCAM-1) in human coronary artery endothelial (HCAE) cells, indicating that microwave fabrication presents a viable and rapid approach to SERS nanoprobe construction. The successful application of SERS nanoprobes to localize biomarker expression in vitro may ultimately be used for early diagnostic and preventative functions in medicine.

Microwave-assisted synthesis of surface-enhanced Raman scattering nanoprobes for cellular sensing.

Surface plasmon resonance (SPR) is a popular technique that allows for sensitive, specific, label-free and real-time assessment of biomolecular interactions. SPR is a nondestructive, modular and flexible tool for various applications in biomedical sciences ranging from cell sorting, cell surface characterization and drug discovery. In this review, we will discuss more specifically how SPR is used to monitor the dynamics of various types of cellular binding events and morphological adherence changes in response to external stimuli.

Article	Year
General, high-affinity approach for the synthesis of fluorophore appended protein nanoparticle assemblies. Chemical communications (Cambridge, England)\| PubMed ID: 15928773	2005
A modular nanoparticle-based system for reagentless small molecule biosensing. Journal of the American Chemical Society\| PubMed ID: 16131178	2005
Selective, reversible, reagentless maltose biosensing with core-shell semiconducting nanoparticles. The Analyst\| PubMed ID: 16440087	2006
Metallothioneins initiate semiconducting nanoparticle cellular toxicity. Small (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 17193582	2006
Prodrugs in cardiovascular therapy. Molecules (Basel, Switzerland)\| PubMed ID: 18560335	2008
Imaging and organelle distribution of fluorescent InGaP/ZnS nanoparticles in glial cells. Nanomedicine (London, England)\| PubMed ID: 19839811	2009
Designed biointerface using near-infrared quantum dots for ultrasensitive surface plasmon resonance imaging biosensors. Analytical chemistry\| PubMed ID: 21604742	2011
Self-assembled multifunctional nanoplexes for gene inhibitory therapy. Nanomedicine (London, England)\| PubMed ID: 21718177	2011
Functionalization of gadolinium metallofullerenes for detecting atherosclerotic plaque lesions by cardiovascular magnetic resonance. Journal of cardiovascular magnetic resonance : official journal of the Society for Cardiovascular Magnetic Resonance\| PubMed ID: 23324435	2013
Zeptomole detection of C-reactive protein in serum by a nanoparticle amplified surface plasmon resonance imaging aptasensor. Scientific reports\| PubMed ID: 24875139	2014
Microwave-assisted synthesis of surface-enhanced Raman scattering nanoprobes for cellular sensing. Colloids and surfaces. B, Biointerfaces\| PubMed ID: 25179113	2014
Surface plasmon resonance: a label-free tool for cellular analysis. Nanomedicine (London, England)\| PubMed ID: 26080702	2015

Marinella G. Sandros

Biography

JoVE Journal Publications

Other Publications