Dynamic multispectral detection of bacteria with nanoplasmonic markers.

Culture-based diagnosis of bacterial diseases is a time-consuming technique that can lead not only to antibiotic resistance or bacterial mutation but also to fast-spreading diseases. Such mutations contribute to the fast deterioration of the patient's health and in some cases the death depending on the complexity of the infection. There is great interest in developing widely available molecular-level diagnostics that provide accurate and rapid diagnosis at the individual level and that do not require sophisticated analysis or expensive equipment. Here, we present a promising analytical approach to detect the presence of pathogenic bacteria based on their dynamic properties enhanced with nanoplasmonic biomarkers. These markers have shown greater photostability and biocompatibility compared to fluorescent markers and quantum dots, and serve as both a selective marker and an amplifying agent in optical biomedical detection. We show that a simple dark-field side- illumination technique can provide sufficiently high-contrast dynamic images of individual plasmonic nanoparticles attached to Escherichia coli (E. coli) for multiplex biodetection. Combined with numerical dynamic filtering, our proposed system shows great potential for the deployment of portable commercial devices for rapid diagnostic tests available to physicians in emergency departments, clinics and public hospitals as point-of-care devices.

Porous Au-Ag Nanoparticles from Galvanic Replacement Applied as Single-Particle SERS Probe for Quantitative Monitoring.

Plasmonic nanostructures have raised the interest of biomedical applications of surface-enhanced Raman scattering (SERS). To improve the enhancement and produce sensitive SERS probes, porous Au-Ag alloy nanoparticles (NPs) are synthesized by dealloying Au-Ag alloy NP-precursors with Au or Ag core in aqueous colloidal environment through galvanic replacement reaction. The novel designed core-shell Au-Ag alloy NP-precursors facilitate controllable synthesis of porous nanostructure, and dealloying degree during the reaction has significant effect on structural and spectral properties of dealloyed porous NPs. Narrow-dispersed dealloyed NPs are obtained using NPs of Au/Ag ratio from 10/90 to 40/60 with Au and Ag core to produce solid core@porous shell and porous nanoshells, having rough surface, hollowness, and porosity around 30-60%. The clean nanostructure from colloidal synthesis exhibits a redshifted plasmon peak up to near-infrared region, and the large accessible surface induces highly localized surface plasmon resonance and generates robust SERS activity. Thus, the porous NPs produce intensely enhanced Raman signal up to 68-fold higher than 100 nm AuNP enhancement at single-particle level, and the estimated Raman enhancement around 7800, showing the potential for highly sensitive SERS probes. The single-particle SERS probes are effectively demonstrated in quantitative monitoring of anticancer drug Doxorubicin release.

Multiplexed Plasmonic Nano-Labeling for Bioimaging of Cytological Stained Samples.

Reliable cytopathological diagnosis requires new methods and approaches for the rapid and accurate determination of all cell types. This is especially important when the number of cells is limited, such as in the cytological samples of fine-needle biopsy. Immunoplasmonic-multiplexed- labeling may be one of the emerging solutions to such problems. However, to be accepted and used by the practicing pathologists, new methods must be compatible and complementary with existing cytopathology approaches where counterstaining is central to the correct interpretation of immunolabeling. In addition, the optical detection and imaging setup for immunoplasmonic-multiplexed-labeling must be implemented on the same cytopathological microscope, not interfere with standard H&E imaging, and operate as a second easy-to-use imaging method. In this article, we present multiplex imaging of four types of nanoplasmonic markers on two types of H&E-stained cytological specimens (formalin-fixed paraffin embedded and non-embedded adherent cancer cells) using a specially designed adapter for SI dark-field microscopy. The obtained results confirm the effectiveness of the proposed optical method for quantitative and multiplex identification of various plasmonic NPs, and the possibility of using immunoplasmonic-multiplexed-labeling for cytopathological diagnostics.

Antibody-Functionalized Gold Nanostar-Mediated On-Resonance Picosecond Laser Optoporation for Targeted Delivery of RNA Therapeutics.

The rapid advances of genetic and genomic technology indicate promising therapeutic potential of genetic materials for regulating abnormal gene expressions causing diseases and disorders. However, targeted intracellular delivery of RNA therapeutics still remains a major challenge hindering the clinical translation. In this study, an elaborated plasmonic optoporation approach is proposed to efficiently and selectively transfect specific cells. The site-specific optoporation is obtained by tuning the spectral range of a supercontinuum pulsed picosecond laser in order for each individual cell binding gold nanostar with their unique resonance peak to magnify the local field strength in the near-infrared region and facilitate a selective delivery of small interfering RNA, messenger RNA, and Cas9-ribonucleoprotein into human retinal pigment epithelial cells. Numerical simulations indicate that optoporation is not due to a plasma-mediated process but rather due to a highly localized temperature rise both in time (few nanoseconds) and space (few nanometers). Taking advantage of the numerical simulation and fine-tuning of the optical strategy, the perforated lipid bilayer of targeted cells undergoes a membrane recovery process, important to retain their viability. The results signify the prospects of antibody functionalized nanostar-mediated optoporation as a simple and realistic gene delivery approach for future clinical practices.

Single point single-cell nanoparticle mediated pulsed laser optoporation.

This article presents an optical platform for studying the dynamics of nanoparticle assisted pulsed laser optoporation of individual living cells. Here plasmonic nanoparticles (NPs) act as markers of the exact spatial position of living cell membranes and as an enhancer for localized pulsed laser perforation. High contrast NP imaging using reflected light microscopy (RLM) allows accurate and automatic laser targeting at individual NPs for spatially controlled laser optoporation of single cells at a single point. The NP imaging method is compatible with fluorescence microscopy and a cellular incubator that allows study of real-time perforation kinetics of live cells and the optomechanical interaction of NPs with membranes. These parameters are of great interest for the development and experimental implementation of the technology of pulsed laser optoporation and transfection applied to single living cells as well as to bulk-level assays.

Designable nanoplasmonic biomarkers for direct microscopy cytopathology diagnostics.

Direct microscopy interpretation of fine-needle biopsy cytological samples is routinely used by practicing cytopathologists. Adding possibility to identify selective and multiplexed biomarkers on the same samples and with the same microscopy technique can greatly improve diagnostic accuracy. In this article, we propose to use biomarkers based on designable plasmonic nanoparticles (NPs) with unique optical properties and excellent chemical stability that can satisfy the above-mentioned requirements. By finely controlling the size and composition of gold-silver alloy NPs and gold nanorods, the NPs plasmonic resonance properties, such as scattering efficiency and resonance peak spectral position, are adjusted in order to provide reliable identification and chromatic differentiation by conventional direct microscopy. Efficient darkfield NPs imaging is performed by using a novel circular side illumination adaptor that can be easily integrated into any microscopy setup while preserving standard cytopathology visualization method. The efficiency of the proposed technology for fast visual detection and differentiation of three spectrally distinct NP-markers is demonstrated in different working media, thus confirming the potential application in conventional cytology preparations. It is worth emphasizing that the presented technology does not interfere with standard visualization with immunohistochemical staining, but should rather be considered as a second imaging modality to confirm the diagnostics.

Cost-effective side-illumination darkfield nanoplasmonic marker microscopy.

We present the development of an innovative technology for quantitative multiplexed cytology analysis based on the application of spectrally distinctive plasmonic nanoparticles (NPs) as optical probes and on cost-effective side-illumination multispectral darkfield microscopy (SIM) as the differential NP imaging method. SIM is based on lateral illumination by arrays of discrete color RGB light emitting diodes (LEDs) of spectrally adjusted plasmonic NPs and consecutive detection by the conventional CMOS color camera. We demonstrate the enhanced contrast and higher resolution of our method for individual NP detection in the liquid medium and of NP markers attached on the cell membrane in a cytology preparation by comparing it to the conventional darkfield microscopy (DFM). The proposed illumination and detection system is compatible with current clinical microscopy equipment used by pathologists and can greatly simplify the adaptation of plasmonic NPs as novel reliable and stable biological multiplexed chromatic markers for biodetection and diagnosis.

In Vivo Laser-Mediated Retinal Ganglion Cell Optoporation Using KV1.1 Conjugated Gold Nanoparticles.

Vision loss caused by retinal diseases affects hundreds of millions of individuals worldwide. The retina is a delicate central nervous system tissue stratified into layers of cells with distinct roles. Currently, there is a void in treatments that selectively target diseased retinal cells, and current therapeutic paradigms present complications associated with off-target effects. Herein, as a proof of concept, we introduce an in vivo method using a femtosecond laser to locally optoporate retinal ganglion cells (RGCs) targeted with functionalized gold nanoparticles (AuNPs). We provide evidence that AuNPs functionalized with an antibody toward the cell-surface voltage-gated K+ channel subunit KV1.1 can selectively deliver fluorescently tagged siRNAs or fluorescein isothiocyanate-dextran dye into retinal cells when irradiated with an 800 nm 100 fs laser. Importantly, neither AuNP administration nor irradiation resulted in RGC death. This system provides a novel, non-viral-based approach that has the potential to selectively target retinal cells in diseased regions while sparing healthy areas and may be harnessed in future cell-specific therapies for retinal degenerative diseases.

3D multiplexed immunoplasmonics microscopy.

Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K(+) channel subunit KV1.1) on human cancer CD44(+) EGFR(+) KV1.1(+) MDA-MB-231 cells and reference CD44(-) EGFR(-) KV1.1(+) 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed technology is simple and compatible with standard epi-fluorescence microscopes used in biological and clinical laboratories. Thus, 3D multiplexed immunoplasmonics microscopy is ready for clinical applications as a cost-efficient alternative to immunofluorescence.

Reflected light microspectroscopy for single-nanoparticle biosensing.

Conventional and dark-field microscopy in the transmission mode is extensively used for single plasmonic nanoparticle (NP) imaging and spectral analysis. However, application of the transmission mode for realtime biosensing to single NP poses strict limitations on the size and material properties of the microfluidic system. This article proposes a simple optical technique based on reflected light microscopy to perform microspectroscopy of a single NP placed in a conventional, nontransparent liquid delivery system. The insertion of a variable spot diaphragm in the optical path reduces the interference effect that occurs at the NP-substrate interface and improves the signal-to-noise ratio in NP imaging. Using this method, we demonstrated spatial imaging and spectral analyses of 60-, 80-, and 100-nm single gold NPs. A single-NP sensor based on a 100-nm NP was used for real-time measurement of bulk refractive index changes in the microfluidic channel and for detection of fast dynamic poly(ethylene glycol) attachment to the NP surface. Finally, electrochemical single-particle microspectroscopy was demonstrated by using a methylene blue electroactive redox tag. The proposed optical approach is expected to significantly improve the miniaturization and multiplexing capabilities of high-throughput biosensing based on single NP.

Electrochemical plasmonic sensing system for highly selective multiplexed detection of biomolecules based on redox nanoswitches.

In this paper, we present the development of a nanoswitch-based electrochemical surface plasmon resonance (eSPR) transducer for the multiplexed and selective detection of DNA and other biomolecules directly in complex media. To do so, we designed an experimental set-up for the synchronized measurements of electrochemical and electro-plasmonic responses to the activation of multiple electrochemically labeled structure-switching biosensors. As a proof of principle, we adapted this strategy for the detection of DNA sequences that are diagnostic of two pathogens (drug-resistant tuberculosis and Escherichia coli) by using methylene blue-labeled structure-switching DNA stem-loop. The experimental sensitivity of the switch-based eSPR sensor is estimated at 5 nM and target detection is achieved within minutes. Each sensor is reusable several times with a simple 8M urea washing procedure. We then demonstrated the selectivity and multiplexed ability of these switch-based eSPR by simultaneously detecting two different DNA sequences. We discuss the advantages of the proposed eSPR approach for the development of highly selective sensor devices for the rapid and reliable detection of multiple molecular markers in complex samples.

Wide-field hyperspectral 3D imaging of functionalized gold nanoparticles targeting cancer cells by reflected light microscopy.

We present a new hyperspectral reflected light microscopy system with a scanned broadband supercontinuum light source. This wide-field and low phototoxic hyperspectral imaging system has been successful for performing spectral three-dimensional (3D) localization and spectroscopic identification of CD44-targeted PEGylated AuNPs in fixed cell preparations. Such spatial and spectral information is essential for the improvement of nanoplasmonic-based imaging, disease detection and treatment in complex biological environment. The presented system can be used for real-time 3D NP tracking as spectral sensors, thus providing new avenues in the spatio-temporal characterization and detection of bioanalytes. 3D image of the distribution of functionalized AuNPs attached to CD44-expressing MDA-MB-231 human cancer cells.

Hyperspectral darkfield microscopy of PEGylated gold nanoparticles targeting CD44-expressing cancer cells.

We present a new hyperspectral darkfield imaging system with a scanned broadband supercontinuum light source. We observed the specific attachment of the functionalized gold plasmonic nanoparticles (AuNPs) targeting CD44(+) human breast cancer cells by conventional and by proposed hyperspectral darkfield microscopy. This wide-field and low phototoxic hyperspectral imaging system has been successful for performing spectral three-dimensional (3D) localization and spectroscopic identification of CD44-targeted PEGylated AuNPs in fixed cell preparations. Such spatial and spectral information is essential for the improvement of nanoplasmonic-based imaging, disease detection and treatment in complex biological environment. Presented system capability for 3D NP tracking will also enable investigation of specific sub-cellular activity with the use of NPs as spectral sensors.

Hyperspectral reflected light microscopy of plasmonic Au/Ag alloy nanoparticles incubated as multiplex chromatic biomarkers with cancer cells.

A hyperspectral microscopy system based on a reflected light method for plasmonic nanoparticle (NP) imaging was designed and compared with a conventional darkfield method for spatial localization and spectroscopic identification of single Au, Ag and Au/Ag alloy NPs incubated with fixed human cancer cell preparations. A new synthesis protocol based on co-reduction of Au and Ag salts combined with the seeded growth technique was used for the fabrication of monodispersed alloy NPs with sizes ranging from 30 to 100 nm in diameter. We validated theoretically and experimentally the performance of 60 nm Au, Ag and Au/Ag (50 : 50) NPs as multiplexed biological chromatic markers for biomedical diagnostics and optical biosensing. The advantages of the proposed reflected light microscopy method are presented for NP imaging in a complex and highly diffusing medium such as a cellular environment. The obtained information is essential for the development of a high throughput, selective and efficient strategy for cancer detection and treatment.

Stability of sputter-deposited gold nanoparticles in imidazolium ionic liquids.

The stability of gold nanoparticles synthesised by sputter deposition has been studied in situ in 1-butyl-3-methylimidazolium ionic liquids with bis(trifluoromethylsulfonyl)imide, tetrafluoroborate, hexafluorophosphate and dicyanamide anions with UV-VIS absorption spectroscopy and transmission electron microscopy. Besides the growth of the gold nanoparticles, two other processes were observed after sputtering, namely aggregation and sedimentation of these nanoparticles. To model the absorption spectra of the sputtered gold nanoparticles, generalized multiparticle Mie calculations were performed. These theoretical calculations confirm the increase in absorbance at longer wavelength for larger aggregates and are in agreement with the experimental observations. It was found that the kinetics of aggregation and sedimentation scale with the viscosity of the ionic liquid. Small amounts of water were found to have a large detrimental influence on the stability of the colloidal suspensions of the gold nanoparticles in ionic liquids. From the large discrepancy between the theoretical and the experimentally observed stability of the NPs, it was concluded that structural forces stabilize the gold nanoparticles. This was also borne out by AFM measurements.

Polarimetric total internal reflection biosensing.

In this paper, a concept of polarimetric total internal reflection (TIR) biosensor based on the method of temporal phase modulation is presented. Measurements of the phase difference between s- and p- polarized light combined with their amplitudes allow simultaneous detection of the bulk refractive index and thickness of the surface biofilms. Obtained experimental sensitivity is better than 10(-5) in terms of refractive index unit and 0.5 nm in biolayer thickness. Relatively simple technological implementation of the TIR sensors on the base of inexpensive and transparent substrates opens a number of novel applications in biosensing and microscopy.

Self-noise-filtering phase-sensitive surface plasmon resonance biosensing.

Emerged as an upgrade of currently available Surface Plasmon Resonance (SPR) biosensing in terms of sensitivity, phase-sensitive SPR technology still requires the minimization of instrumental noises to profit from its projected ultra-low detection limit (10(-8) refractive index units and lower). We present a polarimetry-based methodology for the efficient reduction of main instrumental noises in phase-sensitive measurements. The proposed approach employs a sinusoidal phase modulation of pumping light and is based on selection of proper modulation amplitude and initial phase relation for the first two modulation harmonics (F1 and F2), which enables to subtract amplitude drifts in the difference (F1 - F2) signal while doubling the phase response. The resulting effect can be called self-noise-filtering, since it implies an inherent noise subtraction in every phase sensing measurement. This methodology allows one to tackle drifts related to instabilities of light sources and optical elements and thus drastically lower the detection limit of phase-sensitive SPR sensing even in relatively simple and noisy experimental implementations.

Silicon based total internal reflection bio and chemical sensing with spectral phase detection.

Si-based total internal reflection (TIR) bio/chemical sensor presents an attractive alternative to Surface Plasmon Resonance (SPR) technology due to a relatively simple optical arrangement and technological implementation, as well as a relatively easy bio/chemical immobilization on Si/SiO(2) surface with a number of novel attractive applications. This sensor is based on the control of phase difference between p- and s-polarized components of light reflected from Si/air or Si/water interface in TIR geometry and a high sensitivity of the sensor is granted by a high refractive index of Si (3.56 at 1200 nm). We study properties of TIR sensors in a configuration of spectral phase detection and identify conditions of maximal phase sensitive response. We also experimentally show that the detection limit of Si-based TIR sensor can be lowered down to a level of detection of commercially available SPR devices (10(-6) Refractive Index Units, RIU) under the use of a proper low-noisy method of the phase control. The concept of Si-based TIR opens attractive prospects for the miniaturization of sensor devices, taking advantage of the advanced state of development of Si-based microfabrication technologies, while the proposed spectral phase detection scheme offers much easier packaging and calibration steps.

Phase and amplitude sensitivities in surface plasmon resonance bio and chemical sensing.

We consider amplitude and phase characteristics of light reflected under the Surface Plasmon Resonance (SPR) conditions and study their sensitivities to refractive index changes associated with biological and chemical sensing. Our analysis shows that phase can provide at least two orders of magnitude better detection limit due to the following reasons: (i) Maximal phase changes occur in the very dip of the SPR curve where the vector of probing electric field is maximal, whereas maximal amplitude changes are observed on the resonance slopes: this provides a one order of magnitude larger sensitivity of phase to refractive index variations; (ii) Under a proper design of a detection scheme, phase noises can be orders of magnitude lower compared to amplitude ones, which results in a much better signal-to-noise ratio; (iii) Phase offers much better possibilities for signal averaging and filtering, as well as for image treatment. Applying a phase-sensitive SPR polarimetry scheme and using gas calibration model, we experimentally demonstrate the detection limit of 10(-8) RIU, which is about two orders of magnitude better compared to amplitude-sensitive schemes. Finally, we show how phase can be employed for filtering and treatment of images in order to improve signal-to-noise ratio even in relatively noisy detection schemes. Combining a much better physical sensitivity and a possibility of imaging and sensing in micro-arrays, phase-sensitive methodologies promise a substantial upgrade of currently available SPR technology.