Magneto-Plasmonic Nanoparticle Grid Biosensor with Enhanced Raman Scattering and Electrochemical Transduction for the Development of Nanocarriers for Targeted Delivery of Protected Anticancer Drugs.

Safe administration of highly cytotoxic chemotherapeutic drugs is a challenging problem in cancer treatment due to the adverse side effects and collateral damage to non-tumorigenic cells. To mitigate these problems, promising new approaches, based on the paradigm of controlled targeted drug delivery (TDD), and utilizing drug nanocarriers with biorecognition ability to selectively target neoplastic cells, are being considered in cancer therapy. Herein, we report on the design and testing of a nanoparticle-grid based biosensing platform to aid in the development of new targeted drug nanocarriers. The proposed sensor grid consists of superparamagnetic gold-coated core-shell Fe2Ni@Au nanoparticles, further functionalized with folic acid targeting ligand, model thiolated chemotherapeutic drug doxorubicin (DOX), and a biocompatibility agent, 3,6-dioxa-octanethiol (DOOT). The employed dual transduction method based on electrochemical and enhanced Raman scattering detection has enabled efficient monitoring of the drug loading onto the nanocarriers, attaching to the sensor surface, as well as the drug release under simulated intracellular conditions. The grid's nanoparticles serve here as the model nanocarriers for new TDD systems under design and optimization. The superparamagnetic properties of the Fe2Ni@Au NPs aid in nanoparticles' handling and constructing a dense sensor grid with high plasmonic enhancement of the Raman signals due to the minimal interparticle distance.

Cancer-Targeted Controlled Delivery of Chemotherapeutic Anthracycline Derivatives Using Apoferritin Nanocage Carriers.

The interactions of chemotherapeutic drugs with nanocage protein apoferritin (APO) are the key features in the effective encapsulation and release of highly toxic drugs in APO-based controlled drug delivery systems. The encapsulation enables mitigating the drugs' side effects, collateral damage to healthy cells, and adverse immune reactions. Herein, the interactions of anthracycline drugs with APO were studied to assess the effect of drug lipophilicity on their encapsulation excess n and in vitro activity. Anthracycline drugs, including doxorubicin (DOX), epirubicin (EPI), daunorubicin (DAU), and idarubicin (IDA), with lipophilicity P from 0.8 to 15, were investigated. We have found that in addition to hydrogen-bonded supramolecular ensemble formation with n = 24, there are two other competing contributions that enable increasing n under strong polar interactions (APO(DOX)) or under strong hydrophobic interactions (APO(IDA) of the highest efficacy). The encapsulation/release processes were investigated using UV-Vis, fluorescence, circular dichroism, and FTIR spectroscopies. The in vitro cytotoxicity/growth inhibition tests and flow cytometry corroborate high apoptotic activity of APO(drugs) against targeted MDA-MB-231 adenocarcinoma and HeLa cells, and low activity against healthy MCF10A cells, demonstrating targeting ability of nanodrugs. A model for molecular interactions between anthracyclines and APO nanocarriers was developed, and the relationships derived compared with experimental results.

Advances in Design Strategies of Multiplex Electrochemical Aptasensors.

In recent years, the need for simple, fast, and economical detection of food and environmental contaminants, and the necessity to monitor biomarkers of different diseases have considerably accelerated the development of biosensor technology. However, designing biosensors capable of simultaneous determination of two or more analytes in a single measurement, for example on a single working electrode in single solution, is still a great challenge. On the other hand, such analysis offers many advantages compared to single analyte tests, such as cost per test, labor, throughput, and convenience. Because of the high sensitivity and scalability of the electrochemical detection systems on the one hand and the specificity of aptamers on the other, the electrochemical aptasensors are considered to be highly effective devices for simultaneous detection of multiple-target analytes. In this review, we describe and evaluate multi-label approaches based on (1) metal quantum dots and metal ions, (2) redox labels, and (3) enzyme labels. We focus on recently developed strategies for multiplex sensing using electrochemical aptasensors. Furthermore, we emphasize the use of different nanomaterials in the construction of these aptasensors. Based on examples from the existing literature, we highlight recent applications of multiplexed detection platforms in clinical diagnostics, food control, and environmental monitoring. Finally, we discuss the advantages and disadvantages of the aptasensors developed so far, and debate possible challenges and prospects.

Supramolecular interactions of oxidative stress biomarker glutathione with fluorone black.

Oxidative stress biomarkers, including glutathione (GSH) and related compounds, are involved in a variety of interactions enabling redox potential maintenance in living cells and protection against radicals. Since the oxidative stress is promoting and, in many cases, inducing serious illnesses, monitoring of GSH levels can aid in diagnostics and disease prevention. Herein, we report on the discovery of the formation of a supramolecular ensemble of GSH with fluorone black (9-phenyl fluorone, FB) which is optically active and enables sensitive determination of GSH by resonance elastic light scattering (RELS). We have found that supramolecular interactions of GSH with FB can be probed with spectroscopic, RELS, and electrochemical methods. Our investigations show that RELS intensity for FB solutions increases with GSH concentration while fluorescence emission of FB is not affected, as quenching begins only above 0.8mM GSH. The UV-Vis difference spectra show a positive peak at 383nm and a negative peak at 458nm, indicating a higher-energy absorbing complex in comparison to the non-bonded FB host. Supramolecular interactions of FB with GSH have also been corroborated by electrochemical measurements involving two configurations of FB-GSH ensembles on electrodes: (i) an inverted orientation on Au-coated quartz crystal piezoelectrode (Au@SG-FB), with strong thiolate bonding to gold, and (ii) a non-inverted orientation on glassy carbon electrode (GCE@FB-GS), with weak π-π stacking attachment and efficient charge mediation through the ensemble. The formation of a supramolecular ensemble with hydrogen bonding has also been confirmed by quantum mechanical calculations. The discovery of supramolecular FB-GSH ensemble formation enables elucidating the mechanisms of strong RELS responses, changes in UV-Vis absorption spectra, and the electrochemical reactivity. Also, it provides new insights to the understanding of the efficient charge-transfer in redox potential homeostasis which is likely based on an intermediate formation of a similar type of supramolecular ensembles.

Surface-enhanced Raman scattering investigation of targeted delivery and controlled release of gemcitabine.

Advanced and metastatic cancer forms are extremely difficult to treat and require high doses of chemotherapeutics, inadvertently affecting also healthy cells. As a result, the observed survival rates are very low. For instance, gemcitabine (GEM), one of the most effective chemotherapeutic drugs used for the treatment of breast and pancreatic cancers, sees only a 20% efficacy in penetrating cancer tissue, resulting in <5% survival rate in pancreatic cancer. Here, we present a method for delivering the drug that offers mitigation of side effects, as well as a targeted delivery and controlled release of the drug, improving its overall efficacy. By modifying the surface of gold nanoparticles (AuNPs) with covalently bonded thiol linkers, we have immobilized GEM on the nanoparticle (NP) through a pH-sensitive amide bond. This bond prevents the drug from being metabolized or acting on tissue at physiological pH 7.4, but breaks, releasing the drug at acidic pH, characteristic of cancer cells. Further functionalization of the NP with folic acid and/or transferrin (TF) offers a targeted delivery, as cancer cells overexpress folate and TF receptors, which can mediate the endocytosis of the NP carrying the drug. Thus, through the modification of AuNPs, we have been able to produce a nanocarrier containing GEM and folate/TF ligands, which is capable of targeted controlled-release delivery of the drug, reducing the side effects of the drug and increasing its efficacy. Here, we demonstrate the pH-dependent GEM release, using an ultrasensitive surface-enhanced Raman scattering spectroscopy to monitor the GEM loading onto the nanocarrier and follow its stimulated release. Further in vitro studies with model triple-negative breast cancer cell line MDA-MB-231 have corroborated the utility of the proposed nanocarrier method allowing the administration of high drug doses to targeted cancer cells.

Charge-transfer interactions of Cr species with DNA.

Interactions of Cr species with nucleic acids in living organisms depend strongly on Cr oxidation state and the environmental conditions. As the effects of these interactions range from benign to pre-mutagenic to carcinogenic, careful assessment of the hazard they pose to human health is necessary. We have investigated methods that would enable quantifying the DNA damage caused by Cr species under varying environmental conditions, including UV, O2, and redox potential, using simple instrumental techniques which could be in future combined into a field-deployable instrumentation. We have employed electrochemical quartz crystal nanogravimetry (EQCN), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) to evaluate the extent of DNA damage expressed in terms of guanine oxidation yield (η) and changes in specific characteristics provided by these techniques. The effects of the interactions of Cr species with DNA were analyzed using a model calf thymus DNA (ctDNA) film on a gold electrode (Au@ctDNA) in different media, including: (i) Cr(VI), (ii) Cr(VI) reduced at -0.2V, (iii) Cr(III)+UV radiation+O2, and Cr(III), obtaining the η values: 7.4±1.4, 1.5±0.4, 1.1±0.31%, and 0%, respectively, thus quantifying the hazard posed. The EIS measurements have enabled utilizing the decrease in charge-transfer resistance (Rct) for ferri/ferrocyanide redox probe at an Au@ctDNA electrode to assess the oxidative ctDNA damage by Cr(VI) species. In this case, circular dichroism indicates an extensive damage to the ctDNA hydrogen bonding. On the other hand, Cr(III) species have not induced any damage to ctDNA, although the EQCN measurements show an electrostatic binding to DNA.

Plasmonic nanocarrier grid-enhanced Raman sensor for studies of anticancer drug delivery.

Targeted drug delivery systems using nanoparticle nanocarriers offer remarkable promise for cancer therapy by discriminating against devastating cytotoxicity of chemotherapeutic drugs to healthy cells. To aid in the development of new drug nanocarriers, we propose a novel plasmonic nanocarrier grid-enhanced Raman sensor which can be applied for studies and testing of drug loading onto the nanocarriers, attachment of targeting ligands, dynamics of drug release, assessment of nanocarrier stability in biological environment, and general capabilities of the nanocarrier. The plasmonic nanogrid sensor offers strong Raman enhancement due to the overlapping plasmonic fields emanating from the nearest-neighbor gold nanoparticle nanocarriers and creating the enhancement "hot spots". The sensor has been tested for immobilization of an anticancer drug gemcitabine (2',2'-difluoro-2'-deoxycytidine, GEM) which is used in treatment of pancreatic tumors. The drawbacks of currently applied treatment include high systemic toxicity, rapid drug decay, and low efficacy (ca. 20%). Therefore, the development of a targeted GEM delivery system is highly desired. We have demonstrated that the proposed nanocarrier SERS sensor can be utilized to investigate attachment of targeting ligands to nanocarriers (attachment of folic acid ligand recognized by folate receptors of cancer cells is described). Further testing of the nanocarrier SERS sensor involved drug release induced by lowering pH and increasing GSH levels, both occurring in cancer cells. The proposed sensor can be utilized for a variety of drugs and targeting ligands, including those which are Raman inactive, since the linkers can act as the Raman markers, as illustrated with mercaptobenzoic acid and para-aminothiophenol.

Nanostructured SERS-electrochemical biosensors for testing of anticancer drug interactions with DNA.

Widely used anti-cancer treatments involving chemotherapeutic drugs result in cancer cell damage due to their strong interaction with DNA. In this work, we have developed laboratory biosensors for screening chemotherapeutic drugs and to aid in the assessment of DNA modification/damage caused by these drugs. The sensors utilize surface-enhanced Raman scattering (SERS) spectroscopy and electrochemical methods to monitor sensory film modification and observe the drug-DNA reactivity. The self-assembled monolayer protected gold-disk electrode (AuDE) was coated with a reduced graphene oxide (rGO), decorated with plasmonic gold-coated Fe2Ni@Au magnetic nanoparticles functionalized with double-stranded DNA (dsDNA), a sequence of the breast cancer gene BRCA1. The nanobiosensors AuDE/SAM/rGO/Fe2Ni@Au/dsDNA were then subjected to the action of a model chemotherapeutic drug, doxorubicin (DOX), to assess the DNA modification and its dose dependence. The designed novel nanobiosensors offer SERS/electrochemical transduction, enabling chemically specific and highly sensitive analytical signals generation. The SERS measurements have corroborated the DOX intercalation into the DNA duplex whereas the electrochemical scans have indicated that the DNA modification by DOX proceeds in a concentration dependent manner, with limit of detection LOD=8 µg/mL (S/N=3), with semilog linearity over 3 orders of magnitude. These new biosensors are sensitive to agents that interact with DNA and facilitate the analysis of functional groups for determination of the binding mode. The proposed nanobiosensors can be applied in the first stage of the drug development for testing the interactions of new drugs with DNA before the drug efficacy can be assessed in more expensive testing in vitro and in vivo.

Surface Enhanced Raman Scattering Detection of Cancer Biomarkers with Bifunctional Nanocomposite Probes.

This report describes new findings of an investigation of a bifunctional nanocomposite probe for the detection of cancer biomarkers, demonstrating the viability of magnetic focusing and SERS detection in a microfluidic platform. The nanocomposite probe consists of a magnetic nickel-iron core and a gold shell. Upon bioconjugation, the nanoprobes are magnetically focused on a specific spot in a microfluidic channel, enabling an enrichment of "hot spots" for surface enhanced Raman scattering detection of the targeted carcinoembryonic antigen. The detection sensitivity, with a limit of detection of ∼0.1 pM, is shown to scale with the magnetic focusing time and the nanoparticle size. The latter is also shown to exhibit an excellent agreement between the experimental data and the theoretical simulation. Implications of the findings to the development of rapid and sensitive microfluidic detection of cancer biomarkers are also discussed.

Chromium(VI) but not chromium(III) species decrease mitoxantrone affinity to DNA.

Binding of mitoxantrone (MXT) to double-stranded DNA has been investigated as a model drug-DNA binding system to evaluate the effects of various forms of chromium on the binding properties. We have found that Cr(III), which binds strongly to DNA, does not affect the MXT affinity to DNA. In contrast, Cr(VI), in the form of chromate ions CrO(4)(2-), decreases the MXT affinity to DNA despite electrostatic repulsions with phosphate-deoxyribose chains of DNA. The MXT-DNA binding constant was found to decrease from (1.96 ± 0.005) × 10(5) to (0.77 ± 0.018) × 10(5) M(-1) for Cr(VI) concentration changing from 0 to 30 µM. The influence of Cr(VI) on MXT-DNA binding has been attributed to the oxidation of guanine residue, thus interrupting the intercalation of MXT into the DNA double helix at the preferential CpG intercalation site. This supposition is corroborated by the observed increase in the MXT binding site size from 2 bp (base pairs) to 4-6 bp in the presence of Cr(VI). The measurements of the MXT-DNA binding constant and the MXT binding site size on a DNA molecule have been carried out using spectroscopic, voltammetric, and nanogravimetric techniques, providing useful information on the mechanism of the interactions.

Intervention of glutathione in pre-mutagenic catechol-mediated DNA damage in the presence of copper(II) ions.

The catechol-mediated DNA damage in the presence of Cu(II) ions involves oxidation of guanine to 8-oxoguanine (8-oxoG) and DNA strand scission. It proceeds through the reactive oxygen species (ROS) generation. The mutagenicity of 8-oxoG lesions is due to its miscoding propensity reflected in GC→TA transversion taking place during the DNA repair process. To gain new insights into the nature of catechol-mediated DNA damage and its prevention, we have investigated the changes in DNA melting characteristics and 8-oxoG formation as the indicators of DNA damage in a model calf-thymus DNA system. A novel fluorescence method for DNA melting temperature determination, based on DAPI fluorescent-probe staining, has been proposed. The DNA melting-onset temperature has been found to be more sensitive to DNA damage than the standard melting temperature due to the increased width of the melting transition observed in oxidatively damaged DNA. We have found that the efficiency of Fenton cascade in generating DNA-damaging ROS is higher for catechol than for GSH, two strong antioxidants, mainly due to the much longer distance between ROS-generating radical group in GS to nucleobases than that of semiquinone radical group to nucleobases (2.1nm vs. 0.27nm), making the ROS transport from GSH an order of magnitude less likely to damage DNA because of short lifetime of HO radicals. The antioxidant and DNA-protecting behaviors of GSH have been elucidated. We have found that the redox potential of GSH/GSSG couple is lower than that of catechol/semiquinone couple. Hence, GSH keeps catechol in the reduced state, thereby shutting down the initial step of the catechol-mediated Fenton cascade. The catechol-induced DNA damage in the presence of Cu(II) ions has also been confirmed in studies of ON-OFF hairpin-oligonucleotide beacons.

Mercury/homocysteine ligation-induced ON/OFF-switching of a T-T mismatch-based oligonucleotide molecular beacon.

A molecular beacon (MB) with stem-loop (hairpin) DNA structure and with attached fluorophore-quencher pair at the ends of the strand has been applied to study the interactions of Hg(2+) ions with a thymine-thymine (T-T) mismatch in Watson-Crick base-pairs and the ligative disassembly of MB·Hg(2+) complex by Hg(2+) sequestration with small biomolecule ligands. In this work, a five base-pair stem with configuration 5'-GGTGG...CCTCC-3' for self-hybridization of MB has been utilized. In this configuration, the four GC base-pair binding energy is not sufficient to hybridize fully at intermediate temperatures and to form a hairpin MB conformation. The T-T mismatch built-in into the stem area can effectively bind Hg(2+) ions creating a bridge, T-Hg-T. We have found that the T-Hg-T bridge strongly enhances the ability of MB to hybridize, as evidenced by an unusually large MB melting temperature shift observed on bridge formation, ΔT(m) = +15.1 ± 0.5 °C, for 100 nM MB in MOPS buffer. The observed ΔT(m) is the largest of the ΔT(m) found for other MBs and dsDNA structures. By fitting the parameters of the proposed model of reversible MB interactions to the experimental data, we have determined the T-Hg-T bridge formation constant at 25 °C, K(1) = 8.92 ± 0.42 × 10(17) M(-1) from mercury(II) titration data and K(1) = 1.04 ± 0.51 × 10(18) M(-1) from the bridge disassembly data; ΔG° = -24.53 ± 0.13 kcal/mol. We have found that the biomarker of oxidative stress and cardiovascular disease, homocysteine (Hcys), can sequester Hg(2+) ions from the T-Hg-T complex and withdraw Hg(2+) ions from MB in the form of stable Hg(Hcys)(2)H(2) complexes. Both the model fitting and independent (1)H NMR results on the thymidine-Hg-Hcys system indicate also the high importance of 1:1 complexes. The high value of K(1) for T-Hg-T bridge formation enables analytical determinations of low concentrations of Hg(2+) (limit of detection LOD = 19 nM or 3.8 ppb, based on 3σ method) and Hcys (LOD = 23 nM, 3σ method). The conditional stability constants for Hg(Hcys)H(2)(2+) and Hg(Hcys)(2)H(2) at 52 °C have been determined, ß(112) = 5.37 ± 0.3 × 10(46) M(-3), ß(122) = 3.80 ± 0.6 × 10(68) M(-4), respectively.

Effect of buried potential barrier in label-less electrochemical immunodetection of glutathione and glutathione-capped gold nanoparticles.

The influence of potential barriers, introduced to the immunoglobulin-based sensory films, on voltammetric signals of a redox ion probe has been investigated. Films with positive and negative barriers have been examined by depositing charged self-assembled thiol monolayers as the basal layers of a sensory film. The studies performed with monoclonal anti-glutathione antibody-based sensors using ferricyanide ion probe have shown stronger sensor response to the layer components, as well as to the glutathione-capped gold nanoparticles acting as the antigen, for films with positive potential barrier buried deep in the film than for negative barrier films. The larger changes in differential resistance, peak separation and peak heights observed for films with positive barrier have been attributed to different depth and width of the charge distributions in these films. A buried positive barrier with narrow charge distribution width provides the best conditions for film stability and prevents fouling (less ion-exchanges with the medium). This conclusion has been confirmed by calculations of the electric field distribution and potential profiles in immunosensing films performed by numerical integration of Poisson equation for Gaussian distributions of fixed charges of covalently bound components. The proposed fixed-charge model can aid in rapid evaluation of sensory films in sensor development work. The implications of potential barriers in sensory film design are discussed.

Double-shell gold nanoparticle-based DNA-carriers with poly-L-lysine binding surface.

In view of the prospective applications of polyamine coatings in functional gold nanoparticles for use as carriers in gene delivery systems, in tissue repair and as bactericidal and virucidal non-toxic vehicle, we have investigated the interactions of poly-l-lysine (PLL) with gold nanoparticles (AuNP). Since direct binding of PLL to AuNP is not strong at neutral pH, we have focused on PLL interactions with carboxylated self-assembled monolayers (SAM) on AuNP, such as the citrate-capped AuNP. The double-shell nanoparticles AuNP@Cit/PLL thus produced do not contain any toxic thiols. We have observed strong electrostatic interactions between polycationic chains of PLL and AuNP@Cit in weakly acidic to weakly alkaline solutions (pH 5-9), as evidenced by the bathochromic shift of the local surface plasmon (SP) band and strong increase in resonance elastic light scattering (RELS) intensity. The stoichiometry of interactions evaluated on the basis of RELS data indicates on a hyper-Langmuirian type of interactions with stoichiometric coefficient n = 1.35 (PLL : AuNP@Cit). From the RELS titration data, a shift of the deprotonation constant for the bound PLL has been determined (pK(a) = 11.6 for the bound PLL vs. 10.48 for the free PLL). The deprotonation of PLL leads to AuNP aggregate disassembly, evidenced by sharp RELS decline and hypsochromic shift of SP band. We have found that under these conditions, a residual aggregation due to the interparticle interactions between ß-sheets of PLL overcoat become predominant. The molecular dynamics simulations indicate that multiple hydrogen bonds can also be formed between the PLL linker and the shell molecules of AuNP@Cit. The double-shell nanoparticles, AuNP@Cit/PLL, have been shown to attract DNA molecules using highly sensitive RELS measurements presenting the proof-of-concept for the suitability of this non-toxic nanostructured material for gene delivery applications. The advantage of the proposed material is no toxicity related to the ligand release in gene delivery processes in contrast to the thiol-functionalized AuNP.

"Molecular beacon"-based fluorescent assay for selective detection of glutathione and cysteine.

We report on the development of a fluorescence turn-on "molecular beacon" probe for the detection of glutathione (GSH) and cysteine (Cys). The method is based on a competitive ligation of Hg(2+) ions by GSH/Cys and thymine-thymine (T-T) mismatches in a DNA strand of the self-hybridizing beacon strand. The assay relies on the distance-dependent optical properties of the fluorophore/quencher pair attached to the ends of the molecular beacon DNA strand. In a very selective coordination of Hg(2+) to GSH/Cys, the fluorophore/quencher distance increases concomitantly with the dehybridization and dissociation of the beacon stem T-Hg(2+)-T due to the extraction of Hg(2+) ions. This process results in switching the molecular beacon to the "on" state. The concentration range of the probe is 4-200 nM with the limit of detection (LOD) of 4.1 nM for GSH and 4.2 nM Cys. The probe tested satisfactorily against interference for a range of amino acids including sulfur-containing methionine.

Multimodal coupling of optical transitions and plasmonic oscillations in rhodamine B modified gold nanoparticles.

The optical properties of a photoluminescent dye rhodamine B (RhB) interacting with gold nanoparticles (AuNP) have been investigated using plasmonic absorbance, fluorescence, and resonance elastic light scattering (RELS) spectroscopy. We have found that these interactions result in a multimodal coupling that influence optical transitions in RhB. In absorbance measurements, we have observed for the first time the coupling resulting in strong screening of RhB π-π* transitions, likely caused by a contact adsorption of RhB on a conductive surface of AuNP. The nanoparticles quench also very efficiently the RhB fluorescence. We have determined that the static quenching mechanism with a non-Förster fluorescence resonance energy transfer (FRET) from RhB molecules to AuNP is involved. The Stern-Volmer dependence F(0)/F = f(Q) shows an upward deviation from linearity, attributed to the ultra-high quenching efficiency of AuNP leading to the new extended Stern-Volmer model. A sharp RELS peak of RhB alone (λ(max) = 566 nm) has been observed for the first time and attributed to the resonance fluorescence and enhanced scattering. This peak is completely quenched in the presence of AuNP(22nm). Our quantum mechanical calculations confirm that the distance between AuNP surface and conjugated π-electron system in RhB is well within the range of plasmonic fields extending from AuNP. The optical transition coupling to plasmonic oscillations and the efficient energy transfer due to the interactions of fluorescent dyes with nanoparticles are important for biophysical studies of life processes and applications in nanomedicine.

Resonance elastic light scattering (RELS) spectroscopy of fast non-Langmuirian ligand-exchange in glutathione-induced gold nanoparticle assembly.

The interactions of a biomolecule glutathione (GSH) with citrate-capped gold nanoparticles (AuNP) have been investigated to evaluate the viability of a rapid GSH-capture by gold nanoparticle carriers, as a model system for applications ranging from designing nanoparticle-enhanced functional biosensor interfaces to nanomedicine. The measurements, performed using resonance elastic light scattering (RELS) spectroscopy, have shown a strong dependence of GSH-induced scattering cross-section on gold nanoparticle size. A large increase in RELS intensity after injection of GSH, in a short reaction time (tau=60 s), has been observed for small AuNP (5nm dia.) and ascribed to the fast ligand-exchange followed by AuNP assembly. The unexpected non-Langmuirian concentration dependence of scattering intensity for AuNP (5 nm) indicates on a 2D nucleation and growth mechanism of the ligand-exchange process. The ligand-exchange and small nanoparticle ensemble formation followed by relaxation have been observed in long term (10 h) monitoring of GSH-AuNP interactions by RELS. The results of molecular dynamics and quantum mechanical calculations corroborate the mechanism of the formation of hydrogen-bonded GSH-linkages and interparticle interactions and show that the assembly is driven by multiple H-bonding between GSH-capped AuNP and electrostatic zwitterionic interactions. The RELS spectroscopy has been found as a very sensitive tool for studying interparticle interactions. The application of RELS can be expanded to monitor reactivities and assembly of other monolayer-protected metal clusters, especially in very fast processes which cannot be followed by other techniques.

Nanogravimetric and voltammetric DNA-hybridization biosensors for studies of DNA damage by common toxicants and pollutants.

Electrochemical and nanogravimetric DNA-hybridization biosensors have been developed for sensing single mismatches in the probe-target ssDNA sequences. The voltammetric transduction was achieved by coupling ferrocene moiety to streptavidin linked to biotinylated tDNA. The mass-related frequency transduction was implemented by immobilizing the sensory pDNA on a gold-coated quartz crystal piezoresonators oscillating in the 10MHz band. The high sensitivity of these sensors enabled us to study DNA damage caused by representative toxicants and environmental pollutants, including Cr(VI) species, common pesticides and herbicides. We have found that the sensor responds rapidly to any damage caused by Cr(VI) species, with more severe DNA damage observed for Cr(2)O(7)(2-) and for CrO(4)(2-) in the presence of H(2)O(2) as compared to CrO(4)(2-) alone. All herbicides and pesticides examined caused DNA damage or structural alterations leading to the double-helix unwinding. Among these compounds, paraoxon-ethyl and atrazine caused the fastest and most severe damage to DNA. The physico-chemical mechanism of damaging interactions between toxicants and DNA has been proposed. The methodology of testing voltammetric and nanogravimetric DNA-hybridization biosensors developed in this work can be employed as a simple protocol to obtain rapid comparative data concerning DNA damage caused by herbicide, pesticides and other toxic pollutants. The DNA-hybridization biosensor can, therefore, be utilized as a rapid screening device for classifying environmental pollutants and to evaluate DNA damage induced by these compounds.

Ligand exchange effects in gold nanoparticle assembly induced by oxidative stress biomarkers: homocysteine and cysteine.

The interactions of oxidative stress biomarkers: homocysteine (Hcys) and cysteine (Cys) with the multifunctional gold nanoparticles, important in view of novel biomedical applications in diagnostics and therapy, have been investigated using resonance elastic light scattering (RELS), UV-Vis plasmonic spectroscopy, and high-resolution TEM imaging. The Hcys-induced assembly of gold nanoparticles has been observed for non-ionic surfactant-capped gold nanoparticles as well as for negatively-charged citrate-capped gold nanoparticles. We have observed for the first time the de-aggregation of citrate-capped gold nanoparticle ensembles followed by their conversion to citrate-linked Hcys-capped nanoparticle assemblies. The Cys molecules, which are smaller than Hcys by only one CH(2) group, show much less activity. The mechanisms leading to this intriguing disparity in the abilities of these two thioaminoacids to ligand exchange with surfactant- or citrate-capping molecules of the gold nanoparticle shells are proposed on the basis of the experimental evidence, molecular dynamics simulations, and quantum mechanical calculations. For citrate-capped gold nanoparticles, we postulate the formation of surface complexes facilitated by electrostatic attractions and formation of double hydrogen bonds for both Hcys and Cys. The conformational differences between these two kinds of complexes result in marked differences in the distance between -SH groups of the biomarkers to the gold surface and different abilities to induce nanoparticle assembly. Analytical implications of these mechanistic differences are discussed.

Multifunctional Polypeptide EQCN Sensors: Probing the Cysteamine-Glutathione Film Permeability with Hg(II) Ions.

Multifunctional films are the basis of biosensors and play an important role in the emerging field of nanobioelectronics. In this work, films of a tripeptide glutathione (GSH) immobilized on a self-assembled monolayer of cysteamine (CA-SAM) on a quartz crystal Au piezosensor have been synthesized and characterized using electrochemical quartz crystal nanogravimetry (EQCN) with a Hg(II) ion probe. It has been found that in contrast to previously studied Au/GSH films, the Au/CA-GSH films strongly hinder the formation of Hg° with bulk properties while still allowing for relatively easy permeation by Hg(II) ions. This results in complete disappearance of the sharp Hg° electrodissolution peak which is observed on bare Au and Au/GSH piezosensors. The multiple-peak anodic behavior of Au/CA and bare Au is replaced by a single high-field anodic peak of mercury reoxidation in the case of Au/CA-GSH sensors. The mass-to-charge plots indicate predominant ingress/egress of Hg(II) to/from the film. The strong hindrance of CA-SAM to bulk-Hg° formation is attributed to film-stabilizing formation of surface (CA)2Hg2+ complexes with conformation evaluated by ab initio quantum mechanical calculations of electronic structure using Hartree-Fock methods. The associates CA-GSH provide an additional functionality of the side sulfhydryl group which is free for interactions, e.g. with heavy metals. It is proposed that in the film, the CA-GSH molecules can assume open (extended) conformation or bent hydrogen-bonded conformation with up to four possible internal hydrogen bonds.