Dual-reporter SERS-based biomolecular assay with reduced false-positive signals.

We present a sensitive and quantitative protein detection assay that can efficiently distinguish between specific and nonspecific target binding. Our technique combines dual affinity reagents with surface-enhanced Raman spectroscopy (SERS) and chemometric analysis. We link one Raman reporter-tagged affinity reagent to gold nanoparticles and another to a gold film, such that protein-binding events create a "hot spot" with strong SERS spectra from both Raman reporter molecules. Any signal generated in this context is indicative of recognition by both affinity labels, whereas signals generated by nonspecific binding lack one or the other label, enabling us to efficiently distinguish true from false positives. We show that the number of hot spots per unit area of our substrate offers a quantitative measure of analyte concentration and demonstrate that this dual-label, SERS-linked aptasensor assay can sensitively and selectively detect human α-thrombin in 1% human serum with a limit of detection of 86 pM.

Screening for canine transitional cell carcinoma (TCC) by SERS-based quantitative urine cytology.

Canine lower urinary tract neoplasia is a clinically important disease process that has high mortality due to late stage diagnosis and poorly durable response to treatment. Non-invasive diagnostic techniques (e.g. dipstick test, urine cytology) currently have poor diagnostic value, while more invasive tests (e.g. cystoscopy and biopsy) are costly and often require general anesthesia. We have developed and herein describe a quantitative cytological analysis method based on the use of surface-enhanced Raman spectroscopy (SERS), for identifying cancerous transitional cells in urine using SERS biotags (SBTs) carrying the peptide PLZ4 (amino acid sequence cQDGRMGFc) that targets malignant transitional cells. By analyzing the ratio of the PLZ4-SBTs to an on board control we were able to show that transitional cells had significantly higher ratios (P < 0.05) in patients diagnosed with transitional cell carcinoma (TCC) than in healthy samples.

Biotags Based on Surface-Enhanced Raman Can Be as Bright as Fluorescence Tags.

Surface enhanced Raman spectroscopy (SERS) is a powerful analytical technique that has been proposed as a substitute for fluorescence for biological imaging and detection but is not yet commercially utilized. The reason lies primarily in the lower intensity and poor reproducibility of most metal nanoparticle-based tags as compared to their fluorescence-based counterparts. Here, using a technique that scrupulously preserves the same number of dye molecules in both the SERS and fluorescence measurements, we show that SERS-based biotags (SBTs) with highly reproducible optical properties can be nanoengineered such that their brightness is at least equal to that of fluorescence-based tags.

Advances in superresolution optical fluctuation imaging (SOFI).

We review the concept of superresolution optical fluctuation imaging (SOFI), discuss its attributes and trade-offs (in comparison with other superresolution methods), and present superresolved images taken on samples stained with quantum dots, organic dyes, and plasmonic metal nanoparticles. We also discuss the prospects of SOFI for live cell superresolution imaging and for imaging with other (non-fluorescent) contrasts.

Etchable plasmonic nanoparticle probes to image and quantify cellular internalization.

There is considerable interest in using nanoparticles as labels or to deliver drugs and other bioactive compounds to cells in vitro and in vivo. Fluorescent imaging, commonly used to study internalization and subcellular localization of nanoparticles, does not allow unequivocal distinction between cell surface-bound and internalized particles, as there is no methodology to turn particles 'off'. We have developed a simple technique to rapidly remove silver nanoparticles outside living cells, leaving only the internalized pool for imaging or quantification. The silver nanoparticle (AgNP) etching is based on the sensitivity of Ag to a hexacyanoferrate-thiosulphate redox-based destain solution. In demonstration of the technique we present a class of multicoloured plasmonic nanoprobes comprising dye-labelled AgNPs that are exceptionally bright and photostable, carry peptides as model targeting ligands, can be etched rapidly and with minimal toxicity in mice, and that show tumour uptake in vivo.

Targeted intracellular delivery of proteins with spatial and temporal control.

While a host of methods exist to deliver genetic materials or small molecules to cells, very few are available for protein delivery to the cytosol. We describe a modular, light-activated nanocarrier that transports proteins into cells by receptor-mediated endocytosis and delivers the cargo to the cytosol by light triggered endosomal escape. The platform is based on hollow gold nanoshells (HGN) with polyhistidine tagged proteins attached through an avidity-enhanced, nickel chelation linking layer; here, we used green fluorescent protein (GFP) as a model deliverable cargo. Endosomal uptake of the GFP loaded nanocarrier was mediated by a C-end Rule (CendR) internalizing peptide fused to the GFP. Focused femtosecond pulsed-laser excitation triggered protein release from the nanocarrier and endosome disruption, and the released protein was capable of targeting the nucleoli, a model intracellular organelle. We further demonstrate the generality of the approach by loading and releasing Sox2 and p53. This method for targeting of individual cells, with resolution similar to microinjection, provides spatial and temporal control over protein delivery.

Tough coating proteins: subtle sequence variation modulates cohesion.

Mussel foot protein-1 (mfp-1) is an essential constituent of the protective cuticle covering all exposed portions of the byssus (plaque and the thread) that marine mussels use to attach to intertidal rocks. The reversible complexation of Fe(3+) by the 3,4-dihydroxyphenylalanine (Dopa) side chains in mfp-1 in Mytilus californianus cuticle is responsible for its high extensibility (120%) as well as its stiffness (2 GPa) due to the formation of sacrificial bonds that help to dissipate energy and avoid accumulation of stresses in the material. We have investigated the interactions between Fe(3+) and mfp-1 from two mussel species, M. californianus (Mc) and M. edulis (Me), using both surface sensitive and solution phase techniques. Our results show that although mfp-1 homologues from both species bind Fe(3+), mfp-1 (Mc) contains Dopa with two distinct Fe(3+)-binding tendencies and prefers to form intramolecular complexes with Fe(3+). In contrast, mfp-1 (Me) is better adapted to intermolecular Fe(3+) binding by Dopa. Addition of Fe(3+) did not significantly increase the cohesion energy between the mfp-1 (Mc) films at pH 5.5. However, iron appears to stabilize the cohesive bridging of mfp-1 (Mc) films at the physiologically relevant pH of 7.5, where most other mfps lose their ability to adhere reversibly. Understanding the molecular mechanisms underpinning the capacity of M. californianus cuticle to withstand twice the strain of M. edulis cuticle is important for engineering of tunable strain tolerant composite coatings for biomedical applications.

Modular plasmonic nanocarriers for efficient and targeted delivery of cancer-therapeutic siRNA.

We have combined a versatile and powerful route to deliver nucleic acids with peptide-based cell-specific targeting. siRNA targeting the polo-like kinase gene is in clinical trials for cancer treatment, and here we deliver this RNA selectively to cancer cells displaying the neuropilin-1 epitope using gold nanoshells. Release of the siRNA from the nanoparticles results from irradiation with a pulsed near-infrared laser, which also provides efficient endosomal escape within the cell. As a result, our approach requires 10-fold less material than standard nucleic acid transduction materials and is significantly more efficient than other particle-based methods. We also describe a particle-nucleic acid design that does not rely on modified RNA, thereby making the preparation of these materials more efficient and much less expensive. These improvements, when combined with control over when and where the siRNA is released, could provide the basis for diverse cell biological studies.

Clotting activity of polyphosphate-functionalized silica nanoparticles.

We present a silica nanoparticle (SNP) functionalized with polyphosphate (polyP) that accelerates the natural clotting process of the body. SNPs initiate the contact pathway of the blood-clotting system; short-chain polyP accelerates the common pathway by the rapid formation of thrombin, which enhances the overall blood-clotting system, both by accelerating fibrin generation and by facilitating the regulatory anticoagulation mechanisms essential for hemostasis. Analysis of the clotting properties of bare SNPs, bare polyP, and polyP-functionalized SNPs in plasma demonstrated that the attachment of polyP to SNPs to form polyP-SNPs creates a substantially enhanced synergistic effect that lowers clotting time and increases thrombin production at low concentrations. PolyP-SNP even retains its clotting function at ambient temperature. The polyP-SNP system has the potential to significantly improve trauma-treatment protocols and outcomes in hospital and prehospital settings.

Quantitative ratiometric discrimination between noncancerous and cancerous prostate cells based on neuropilin-1 overexpression.

A multiplexed, ratiometric method is described that can confidently distinguish between cancerous and noncancerous epithelial prostate cells in vitro. The technique is based on bright surface-enhanced resonance Raman scattering (SERRS) biotags (SBTs) infused with unique Raman reporter molecules, and carrying cell-specific peptides. Two sets of SBTs were used. One targets the neuropilin-1 (NRP-1) receptors of cancer cells through the RPARPAR peptide. The other functions as a positive control (PC) and binds to both noncancerous and cancer cells through the HIV-derived TAT peptide. Point-by-point 2D Raman maps of the spatial distribution of the two tags were constructed with subcellular resolution from cells simultaneously incubated with the two sets of SBTs. Averaging the SERRS signal over a given cell yielded an NRP/PC ratio from which a robust quantitative measure of the overexpression of the NRP-1 by the cancer cell line was extracted. The use of a local, on-cell reference produces quantitative, statistically robust measures of overexpression independent of such sources of uncertainty as variations in the location of the focal plane, the local cell concentration, and turbidity.

Mesoporous multifunctional upconversion luminescent and magnetic "nanorattle" materials for targeted chemotherapy.

Nanorattles consisting of hydrophilic, rare-earth-doped NaYF(4) shells each containing a loose magnetic nanoparticle were fabricated through an ion-exchange process. The inner magnetic Fe(3)O(4) nanoparticles are coated with a SiO(2) layer to avoid iron leaching in acidic biological environments. This multifunctional mesoporous nanostructure with both upconversion luminescent and magnetic properties has excellent water dispersibility and a high drug-loading capacity. The material emits visible luminescence upon NIR excitation and can be directed by an external magnetic field to a specific target, making it an attractive system for a variety of biological applications. Measurements on cells incubated with the nanorattles show them to have low cytotoxicity and excellent cell imaging properties. In vivo experiments yield highly encouraging tumor shrinkage with the antitumor drug doxorubicin (DOX) and significantly enhanced tumor targeting in the presence of an applied magnetic field.

A luminescent and biocompatible photoCORM.

The water-soluble rhenium(I) complex fac-[Re(bpy)(CO)(3)(thp)](+) (1) [CF(3)SO(3)(-) salt; bpy = 2,2'-bipyridine, thp = tris(hydroxymethyl)phosphine] is both strongly luminescent and photoactive toward carbon monoxide release. It is stable in aerated aqueous media, is incorporated into cells from the human prostatic carcinoma cell line PPC-1, and shows no apparent cytotoxicity. Furthermore, the solvated Re(I) photoproduct of CO release (2) is also luminescent, a feature that allows one to track the transformation of 1 to 2 inside such cells using confocal fluorescence microscopy. In this context, 1 is a very promising candidate as a photoactivated CO releasing moiety (photoCORM) with potential therapeutic applications.

Gold-Protein Composite Nanoparticles for Enhanced X-ray Interactions: A Potential Formulation for Triggered Release.

Drug-delivery vehicles have been used extensively to modulate the biodistribution of drugs for the purpose of maximizing their therapeutic effects while minimizing systemic toxicity. The release characteristics of the vehicle must be balanced with its encapsulation properties to achieve optimal delivery of the drug. An alternative approach is to design a delivery vehicle that preferentially releases its contents under specific endogenous (e.g., tissue pH) or exogenous (e.g., applied temperature) stimuli. In the present manuscript, we report on a novel delivery system with potential for triggered release using external beam radiation. Our group evaluated Zein protein as the basis for the delivery vehicle and used radiation as the exogenous stimulus. Proteins are known to react with free radicals, produced during irradiation in aqueous suspensions, leading to aggregation, fragmentation, amino acid modification, and proteolytic susceptibility. Additionally, we incorporated gold particles into the Zein protein matrix to create hybrid Zein-gold nanoparticles (ZAuNPs). Zein-only nanoparticles (ZNPs) and ZAuNPs were subsequently exposed to kVp radiation (single dose ranging from 2 to 80 Gy; fractionated doses of 2 Gy delivered 10 times) and characterized before and after irradiation. Our data indicated that the presence of gold particles within Zein particles was correlated with significantly higher levels of alterations to the protein, and was associated with higher rates of release of the encapsulated drug compound, Irinotecan. The aggregate results demonstrated a proof-of-principle that radiation can be used with gold nanoparticles to modulate the release rates of protein-based drug-delivery vehicles, such as ZNPs.

Liposomal OTS964, a TOPK inhibitor: a simple method to estimate OTS964 association with liposomes that relies on enhanced OTS964 fluorescence when bound to albumin.

OTS964 is an inhibitor of T-lymphokine-activated killer cell-originated protein kinase (TOPK), a protein kinase important for mitosis and highly expressed in ovarian and lung cancers. This compound demonstrated potent anti-proliferative activity in a panel of cell lines positive for TOPK; however, when administered to mouse xenograft models, adverse hematopoietic toxicities were observed. To overcome this problem, OTS964 was encapsulated into liposomes and a liposomal formulation of OTS964 is now considered a lead candidate for clinical development. To support clinical development of this formulation, it is critically important to define assays that can easily distinguish between free and liposomal OTS964. Here, we develop a new assay to determine liposomal OTS964 encapsulation (percentage of drug associated with the liposomes) and OTS964 that is dissociated from the liposomes (percentage of drug released from liposomes) by monitoring the enhanced OTS964 fluorescence after its binding to albumin. The optical properties of OTS964 were investigated and three absorbance peaks were identified (235 nm, 291 nm, and 352 nm). Fluorescence was observed at 350 nm (excitation) and 470 nm (emission). Interestingly, the fluorescence of OTS964 increased 18-fold in the presence of serum proteins and more specifically albumin. This phenomenon was used to discriminate between the amounts of drug associated with the liposomes or released from the liposomes. Controls consisting of liposomal OTS964 permeabilized with saponins or octyl glucopyranoside served to confirm that drug release could be monitored by albumin-associated increases in fluorescence. The OTS964 liposomal formulation proved to be very stable with less than 10% release after 4 days in phosphate-buffered saline at 37 °C. The quantity of drug associated with the liposomal surface but not inside the liposomes could also be estimated using this approach. These studies present a novel approach to characterize liposomal release of OTS964, in real time and in a non-invasive manner while acquiring additional information about the spatial distribution of liposomal drug.

Rapid identification by surface-enhanced Raman spectroscopy of cancer cells at low concentrations flowing in a microfluidic channel.

Reliable identification and collection of cells from bodily fluids is of growing interest for monitoring patient response to therapy and for early detection of disease or its recurrence. We describe a detection platform that combines microfluidics with surface-enhanced Raman spectroscopy (SERS) for the identification of individual mammalian cells continuously flowing in a microfluidics channel. A mixture of cancerous and noncancerous prostate cells was incubated with SERS biotags (SBTs) developed and synthesized by us, then injected into a flow-focused microfluidic channel, which forces the cells into a single file. The spectrally rich SBTs are based on a silver nanoparticle dimer core labeled with a Raman-active small reporter molecule paired with an affinity biomolecule, providing a unique barcode whose presence in a composite SERS spectrum can be deconvoluted. Individual cancer cells passing through the focused laser beam were correctly identified among a proportionally larger number of other cells by their Raman signatures. We examine two deconvolution strategies: principal component analysis and classical least-squares. The deconvolution strategies are used to unmix the overall spectrum to determine the relative contributions between two SBT barcodes, where one SBT barcode indicates neuropilin-1 overexpression, while a second SBT barcode is more universal and indicates unspecific binding to a cell's membrane. Highly reliable results were obtained for all of the cell mixture ratios tested, the lowest being 1 in 100 cells.

Light-activated RNA interference in human embryonic stem cells.

We describe a near infrared (NIR) light-activated gene silencing method in undifferentiated human embryonic stem cell (hESC) using a plasmonic hollow gold nanoshell (HGN) as the siRNA carrier. Our modular biotin-streptavidin coupling strategy enables positively charged TAT-peptide to coat oligonucleotides-saturated nanoparticles as a stable colloid formation. TAT-peptide coated nanoparticles with dense siRNA loading show efficient penetration into a wide variety of hESC cell lines. The siRNA is freed from the nanoparticles and delivered to the cytosol by femtosecond pulses of NIR light with potentially exquisite spatial and temporal control. The effectiveness of this approach is shown by targeting GFP and Oct4 genes in undifferentiated hESC (H9). The accelerated expression of differentiation markers for all three germ layers resulting from Oct4 knockdown confirms that this method has no detectable adverse effects that limit the range of differentiation. This biocompatible and NIR laser-activated patterning method makes possible single cell resolution of siRNA delivery for diverse studies in stem cell biology, tissue engineering and regenerative medicine.

Rapid, Reversible Release from Thermosensitive Liposomes Triggered by Near-Infra-Red Light.

We present a novel drug carrier consisting of plasmonic hollow gold nanoshells (HGN) chemically tethered to liposomes made temperature sensitive with lysolipids (LTSL). Continuous-wave irradiation by physiologically friendly near infra-red light at 800 nm for 2.5 minutes at laser intensities an order of magnitude below that known to damage skin generates heating localized to the liposome membrane. The heating increases the liposome permeability in an irradiation dose-dependent, but reversible manner, resulting in rapid release of small molecules such as the self-quenching dye carboxyfluorescein or the chemotherapeutic doxorubicin, without raising the bulk temperature. The local rise in nanoshell temperature under laser irradiation was inferred by comparing dye release rates from the LTSL via bulk heating to that induced by irradiation. Laser-irradiation of LTSL enables precise control of contents release with low temperature gradients confined to areas irradiated by the laser focus. The combined effects of rapid local release and localized hyperthermia provide a synergistic effect as shown by a near doubling of androgen resistant PPC-1 prostate cancer cell toxicity compared to the same concentration of free doxorubicin.