Surface-Enhanced Raman Scattering Optophysiology Nanofibers for the Detection of Heavy Metals in Single Breast Cancer Cells.

Mercury(II) ions (Hg2+) and silver ions (Ag+) are two of the most hazardous pollutants causing serious damage to human health. Here, we constructed surface-enhanced Raman scattering (SERS)-active nanofibers covered with 4-mercaptopyridine (4-Mpy)-modified gold nanoparticles to detect Hg2+ and Ag+. Experimental evidence suggests that the observed spectral changes originate from the combined effect of (i) the coordination between the nitrogen on 4-Mpy and the metal ions and (ii) the 4-Mpy molecular orientation (from flatter to more perpendicular with respect to the metal surface). The relative intensity of a pair of characteristic Raman peaks (at ∼428 and ∼708 cm-1) was used to quantify the metal ion concentration, greatly increasing the reproducibility of the measurement compared to signal-on or signal-off detection based on a single SERS peak. The detection limit of this method for Hg2+ is lower than that for the Ag+ (5 vs 100 nM), which can be explained by the stronger interaction energy between Hg2+ and N compared to Ag+ and N, as demonstrated by density functional theory calculations. The Hg2+ and Ag+ ions can be masked by adding ethylenediaminetetraacetate and Cl-, respectively, to the Hg2+ and Ag+ samples. The good sensitivity, high reproducibility, and excellent selectivity of these nanosensors were also demonstrated. Furthermore, detection of Hg2+ in living breast cancer cells at the subcellular level is possible, thanks to the nanometric size of the herein described SERS nanosensors, allowing high spatial resolution and minimal cell damage.

Branched Au Nanoparticles on Nanofibers for Surface-Enhanced Raman Scattering Sensing of Intracellular pH and Extracellular pH Gradients.

The development of plasmonic-active nanosensors for surface-enhanced Raman scattering (SERS) sensing is important for gaining knowledge on intracellular and extracellular chemical processes, hypoxia detection, and label-free detection of neurotransmitters and metabolites, among other applications in cell biology. The fabrication of SERS nanosensors for optophysiology measurements using substrates such as nanofibers with a uniform distribution of plasmonic nanoparticles (NPs) remains a critical hurdle. We report here on a strategy using block copolymer brush-layer templating and ligand exchange for fabricating highly reproducible and stable SERS-active nanofibers with tip diameters down to 60 nm and covered with well-dispersed and uniformly distributed branched AuNPs, which have intrinsic hotspots favoring inherently high plasmonic sensitivity. Among the SERS sensors investigated, those with Au nanostars with short branches [AuNS(S)s] exhibit the greatest SERS sensitivity, as verified also by COMSOL Multiphysics simulations. Functionalization of the AuNS(S)s with the pH-sensitive molecule, 4-mercaptobenzoic acid, led to SERS nanosensors capable of quantifying pH over a linear range of 6.5-9.5, covering the physiological range. These pH nanosensors were shown to be able to detect the intracellular pH as well as extracellular pH gradients of in vitro breast cancer cells with minimal invasiveness and improved SERS sensitivity, along with a high spatial resolution capability.

Portable and field-deployed surface plasmon resonance and plasmonic sensors.

Plasmonic sensors are ideally suited for the design of small, integrated, and portable devices that can be employed in situ for the detection of analytes relevant to environmental sciences, clinical diagnostics, infectious diseases, food, and industrial applications. To successfully deploy plasmonic sensors, scaled-down analytical devices based on surface plasmon resonance (SPR) and localized surface plasmon resonance (LSPR) must integrate optics, plasmonic materials, surface chemistry, fluidics, detectors and data processing in a functional instrument with a small footprint. The field has significantly progressed from the implementation of the various components in specifically designed prism-based instruments to the use of nanomaterials, optical fibers and smartphones to yield increasingly portable devices, which have been shown for a number of applications in the laboratory and deployed on site for environmental, biomedical/clinical, and food applications. A roadmap to deploy plasmonic sensors is provided by reviewing the current successes and by laying out the directions the field is currently taking to increase the use of field-deployed plasmonic sensors at the point-of-care, in the environment and in industries.

In Vitro Drug Release and Biocatalysis from pH-Responsive Gold Nanoparticles Synthesized Using Doxycycline.

pH-sensitive doxycycline gold nanoparticles (doxy-AuNPs) are reported here to act as effective drug nanocarriers and as biocatalysts. The AuNPs were synthesized with doxy as the reducing and capping agent. Various parameters were optimized to find the best conditions for the synthesis of doxy-AuNPs, and these were characterized with UV-vis, X-ray diffraction (XRD), FTIR, and transmission electron microscopy (TEM). Doxy-AuNPs were then loaded with the anticancer drug doxorubicin (DOX), where 70% of the initially available drug was loaded within 24 h. Furthermore, pH-dependent drug release was measured at 60% with in vitro measurements in phosphate-buffered saline (PBS). In addition, the doxy-AuNPs were applied as a biocatalyst. Oxidation of dopamine was taken as a model reaction to determine the catalytic activity of doxy-AuNPs. Almost complete oxidation of dopamine occurred in 5 min, which indicates the fast response of synthesized doxy-AuNPs as a biocatalyst. Hence, doxy-AuNPs are a versatile platform for drug loading and biocatalyst.

Machine-Learning-Driven Surface-Enhanced Raman Scattering Optophysiology Reveals Multiplexed Metabolite Gradients Near Cells.

The extracellular environment is a complex medium in which cells secrete and consume metabolites. Molecular gradients are thereby created near cells, triggering various biological and physiological responses. However, investigating these molecular gradients remains challenging because the current tools are ill-suited and provide poor temporal and special resolution while also being destructive. Herein, we report the development and application of a machine learning approach in combination with a surface-enhanced Raman spectroscopy (SERS) nanoprobe to measure simultaneously the gradients of at least eight metabolites in vitro near different cell lines. We found significant increase in the secretion or consumption of lactate, glucose, ATP, glutamine, and urea within 20 µm from the cells surface compared to the bulk. We also observed that cancerous cells (HeLa) compared to fibroblasts (REF52) have a greater glycolytic rate, as is expected for this phenotype. Endothelial (HUVEC) and HeLa cells exhibited significant increase in extracellular ATP compared to the control, shining light on the implication of extracellular ATP within the cancer local environment. Machine-learning-driven SERS optophysiology is generally applicable to metabolites involved in cellular processes, providing a general platform on which to study cell biology.

Hybridization conditions of oligonucleotide-capped gold nanoparticles for SPR sensing of microRNA.

MicroRNA (miRNA) sensing, especially the miRNA-200 family, is increasingly targeted for cancer diagnostics. As the sensing schemes often rely on nanoparticles functionalized with a specific oligonucleotide, we investigate the hydribization conditions using the common case of surface plasmon resonance (SPR) sensing of miRNA and a gold nanoparticle (Au NP) competitor. In this type of assays, the Au NPs compete with the microRNA to bind the capture probe immobilized on the gold surface. In our study, we simplify and improve the detection procedure by adopting 11-mercaptoundecanoic acid (11-MUA) as linker to the gold surface, not only omitting the blocking step of 6-mercapto-1-hexanol (MCH), but also increasing the probe density. We report that the response in our SPR sensing studies increased with the size of Au NPs according to the plasmon ruler equation, but the larger AuNPs of 32 nm lacked colloidal stability. In addition, decreasing the ratio of oligonucleotide to Au NPs and the addition of polyethylene glycol (PEG) to hybridization buffer also favored a better response in SPR sensing of miRNA. The optimization led to an improved detection sensitivity in our competition method and a detection limit as low as 500 pM for miRNA-200b without amplification of miRNA and use of other amplification schemes.

Rational Design of Magnetic Micronanoelectrodes for Recognition and Ultrasensitive Quantification of Cysteine Enantiomers.

Driven by the urgent need for recognition and quantification of trace amino acids enantiomers in various biologic samples, we demonstrate for the first time an ultrasensitive electrochemical chiral biosensor for cysteine (Cys) based on magnetic nanoparticles (Fe3O4@PDA/Cu xO) as electrode units. d-Cys-Cu2+-d-Cys formed in the presence of cysteine exhibits strong stability and a shielding effect on the redox current of indicator Cu2+, which can be used to quantify and recognize d-Cys by square wave voltammetry. Simultaneous detection of d-Cys and homocysteine (Hcy) is achieved in the presence of other amino acids, demonstrating an excellent selectivity of the sensor. Moreover, aided by the enrichment treatment effect of magnetic micronanoelectrodes, an ultrahigh sensitivity up to 102 µA µM-1 cm-2 was achieved, the detection limit is reduced to picomolar level (83 pM) for d-Cys and can be used for the recognition of cysteine enantiomers. The proposed method has been verified by real sample analysis with satisfactory results. The results highlight the feasibility of our proposed strategy for magnetic micronanoelectrode sensor, electrochemical recognition, and quantification of d-Cys, which can be more broadly applicable than that with traditional electrode structures and further advance the field of electrochemical sensors.

Novel tungsten phosphide embedded nitrogen-doped carbon nanotubes: A portable and renewable monitoring platform for anticancer drug in whole blood.

Biosensors based on converting the concentration of analytes in complex samples into single electrochemical signals are attractive candidates as low cost, high-throughput, portable and renewable sensor platforms. Here, we describe a simple but practical analytical device for sensing an anticancer drug in whole blood, using the detection of methotrexate (MTX) as a model system. In this biosensor, a novel carbon-based composite, tungsten phosphide embedded nitrogen-doped carbon nanotubes (WP/N-CNT), was fixed to the electrode surface that supported redox cycling. The electronic transmission channel in nitrogen doped carbon nanotubes (N-CNT) and the synergistic effect of uniform distribution tungsten phosphide (WP) ensured that the electrode materials have outstanding electrical conductivity and catalytic performance. Meanwhile, the surface electronic structure also endows its surprisingly reproducible performance. To demonstrate portable operation for MTX sensing, screen printing electrodes (SPE) was modified with WP/N-CNT. The sensor exhibited low detection limits (45 nM), wide detection range (0.01-540 µM), good selectivity and long-term stability for the determination of MTX. In addition, the technique was successfully applied for the determination of MTX in whole blood.

Tracking Silent Hypersensitivity Reactions to Asparaginase during Leukemia Therapy Using Single-Chip Indirect Plasmonic and Fluorescence Immunosensing.

Microbial asparaginase is an essential component of chemotherapy for the treatment of childhood acute lymphoblastic leukemia (cALL). Silent hypersensitivity reactions to this microbial enzyme need to be monitored accurately during treatment to avoid adverse effects of the drug and its silent inactivation. Here, we present a dual-response anti-asparaginase sensor that combines indirect SPR and fluorescence on a single chip to perform ELISA-type immunosensing, and correlate measurements with classical ELISA. Analysis of serum samples from children undergoing cALL therapy revealed a clear correlation between single-chip indirect SPR/fluorescence immunosensing and ELISA used in clinical settings (R2 > 0.9). We also report that the portable SPR/fluorescence system had a better sensitivity than classical ELISA to detect antibodies in clinical samples with low antigenicity. This work demonstrates the reliability of dual sensing for monitoring clinically relevant antibody titers in clinical serum samples.

Correction: Ultra-low fouling alkylimidazolium modified surfaces for the detection of HER2 in breast cancer cell lysates.

Correction for 'Ultra-low fouling methylimidazolium modified surfaces for the detection of HER2 in breast cancer cell lysates' by Alexandra Aubé et al., Analyst, 2017, 142, 2343-2353.

Surface Plasmon Resonance Clinical Biosensors for Medical Diagnostics.

The design and application of sensors for monitoring biomolecules in clinical samples is a common goal of the sensing research community. Surface plasmon resonance (SPR) and other plasmonic techniques such as localized surface plasmon resonance (LSPR) and imaging SPR are reaching a maturity level sufficient for their application in monitoring biomolecules in clinical samples. In recent years, the first examples for monitoring antibodies, proteins, enzymes, drugs, small molecules, peptides, and nucleic acids in biofluids collected from patients afflicted with a series of medical conditions (Alzheimer's, hepatitis, diabetes, leukemia, and cancers such as prostate and breast cancers, among others) demonstrate the progress of SPR sensing in clinical chemistry. This Perspective reviews the current status of the field, showcasing a series of early successes in the application of SPR for clinical analysis and detailing a series of considerations regarding sensing schemes, exposing issues with analysis in biofluids, and comparing SPR with ELISA, while providing an outlook of the challenges currently associated with plasmonic materials, instrumentation, microfluidics, bioreceptor selection, selection of a clinical market, and validation of a clinical assay for applying SPR sensors to clinical samples. Research opportunities are proposed to further advance the field and transition SPR biosensors from research proof-of-concept stage to actual clinical applications.

Ultra-low fouling alkylimidazolium modified surfaces for the detection of HER2 in breast cancer cell lysates [corrected].

We synthesized novel ultra-low fouling ionic liquids and demonstrated their use with surface plasmon resonance (SPR) sensing for the analysis of HER2 in breast cancer cell lysates. Whilst biomarkers are commonly detected in serum, this remains challenging for cancer diagnosis due to their low concentrations in circulation and in some cases, there is a poor correlation between serum and tissue concentrations. Therefore, a cell lysate constitutes an interesting biosample for cancer diagnosis and typing, which has been largely unexploited for chemical biosensing of cancer biomarkers. However, high fouling of surfaces in contact with the cell lysate and the absence of effective surface chemistry to prevent fouling are currently limiting biomarker analysis in cell lysates. To address this challenge, we report the synthesis of 1-(carboxyalkyl)-3-(12-mercaptododecyl)-1H-imidazolium ionic liquids with different anions (Br-, BF4-, PF6-, ClO4-, and NTf2-) and ethyl and pentyl chains to form monolayers and analyse specific proteins from cell lysates. The most efficient ionic liquid monolayer, 1-(carboxyethyl)-3-(12-mercaptododecyl)-1H-imidazolium bromide, was able to eliminate the nonspecific adsorption (surface coverage of 2 ± 2 ng cm-2) of a concentrated cell lysate (protein concentration of ∼3.5 mg mL-1), which was significantly better than carboxy-PEG (surface coverage of 14 ± 7 ng cm-2), a benchmark monolayer commonly used to reduce nonspecific adsorption. These ionic liquid monolayers were modified with anti-HER2 and the detection of the HER2 breast cancer biomarker was carried out in crude breast cancer cell lysates, as shown with HER2-negative MCF-7 cells spiked with HER2 and with HER2 positive SK-BR-3 cells.

Epstein-Barr virus-induced gene 3 (EBI3) can mediate IL-6 trans-signaling.

Epstein-Barr virus-induced gene 3 (EBI3) is a subunit of the composite cytokines IL-27 and IL-35. Both have beneficial functions or effects in models of infectious and autoimmune diseases. This suggests that administration of EBI3 could be therapeutically useful by binding free p28 and p35 to generate IL-27 and IL-35. IL-27- and IL-35-independent functions of EBI3 could compromise its therapeutic uses. We therefore assessed the effects of EBI3 on cytokine receptor-expressing cells. We observed that EBI3 activates STAT3 and induces the proliferation of the IL-6-dependent B9 mouse plasmacytoma cell line. Analyses using blocking mAbs and Ba/F3 transfectants expressing gp130 indicate that EBI3 activity was linked to its capacity to mediate IL-6 trans-signaling, albeit less efficiently than soluble IL-6Rα. In line with this interpretation, co-immunoprecipitation and SPR experiments indicated that EBI3 binds IL-6. An important pro-inflammatory function of IL-6 trans-signaling is to activate blood vessel endothelial cells. We observed that EBI3 in combination with IL-6 could induce the expression of chemokines by human venal endothelial cells. Our results indicate that EBI3 can promote pro-inflammatory IL-6 functions by mediating trans-signaling. These unexpected observations suggest that use of EBI3 as a therapeutic biologic for autoimmune diseases will likely require co-administration of soluble gp130 to prevent the side effects associated with IL-6 trans-signaling. Together with previous studies that demonstrated activation of IL-6R by p28 (IL-30), new findings further suggest a complex interrelation between IL-27 and IL-6.

Development of Escherichia coli Asparaginase II for Immunosensing: A Trade-Off between Receptor Density and Sensing Efficiency.

The clinical success of Escherichia colil-asparaginase II (EcAII) as a front line chemotherapeutic agent for acute lymphoblastic leukemia (ALL) is often compromised because of its silent inactivation by neutralizing antibodies. Timely detection of silent immune response can rely on immobilizing EcAII, to capture and detect anti-EcAII antibodies. Having recently reported the use of a portable surface plasmon resonance (SPR) sensing device to detect anti-EcAII antibodies in undiluted serum from children undergoing therapy for ALL (Aubé et al., ACS Sensors2016, 1 (11), 1358-1365), here we investigate the impact of the quaternary structure and the mode of immobilization of EcAII onto low-fouling SPR sensor chips on the sensitivity and reproducibility of immunosensing. We show that the native tetrameric structure of EcAII, while being essential for activity, is not required for antibody recognition because monomeric EcAII is equally antigenic. By modulating the mode of immobilization, we observed that low-density surface coverage obtained upon covalent immobilization allowed each tetrameric EcAII to bind up to two antibody molecules, whereas high-density surface coverage arising from metal chelation by N- or C-terminal histidine-tag reduced the sensing efficiency to less than one antibody molecule per tetramer. Nonetheless, immobilization of EcAII by metal chelation procured up to 10-fold greater surface coverage, thus resulting in increased SPR sensitivity and allowing reliable detection of lower analyte concentrations. Importantly, only metal chelation achieved highly reproducible immobilization of EcAII, providing the sensing reproducibility that is required for plasmonic sensing in clinical samples. This report sheds light on the impact of multiple factors that need to be considered to optimize the practical applications of plasmonic sensors.

Biosensors and nanobiosensors for therapeutic drug and response monitoring.

Therapeutic drug monitoring (TDM) is required for pharmaceutical drugs with dosage limitations or toxicity issues where patients undergoing treatment with these drugs require frequent monitoring. This allows for the concentration of such pharmaceutical drugs in a patient's biofluid to be closely monitored in order to assess the pharmacokinetics, which could result in an adjustment of dosage or in medical intervention if the situation becomes urgent. Biosensors are a class of analytical techniques competent in the rapid quantification of therapeutic drugs and recent developments in instrumental platforms and in sensing schemes, as well as the emergence of nanobiosensors, have greatly contributed to the principal examples of these sensors for therapeutic drug monitoring. Based on initial success stories, it is clear that (nano)biosensors could pave the way for therapeutic drug monitoring of many commonly administered drugs and for new drugs that will be introduced to the market allowing for safe and optimal dosing across a wide range of pharmaceuticals. In this review, we report on the recent developments in biosensing and nanobiosensing techniques and, focussing mainly on anti-cancer agents and antibiotics, we discuss the different classes of molecules upon which therapeutic drug monitoring has already been successfully applied. The potential contributions of (nano)biosensors are also reviewed for the emerging areas of therapeutic response monitoring, where markers are monitored to ensure compliance of a patient to a treatment and in the area of cellular response to therapeutic drugs in order to identify cytotoxic effects of drugs on cells or to identify patients responding to a drug.

Miniature multi-channel SPR instrument for methotrexate monitoring in clinical samples.

A multi-channel fully integrated SPR biosensor was applied for the analysis of an anti-cancer drug, methotrexate (MTX) as a potential analytical tool used in clinical chemistry laboratories for therapeutic drug monitoring (TDM). MTX concentrations in a patient's serum undergoing chemotherapy treatments can be determined by surface plasmon resonance (SPR) sensing using folic acid-functionalized gold nanoparticles (FA-AuNP) in competition with MTX for the bioreceptor, human dihydrofolate reductase (hDHFR) immobilized on the SPR sensor chip. To validate this biosensor, 13 nm FA-AuNP were shown to interact with immobilized hDHFR in the absence of MTX and this interaction was inhibited in the presence of MTX. The sensor was calibrated for MTX in phosphate buffer at different dynamic range by varying nanoparticle sizes (5, 13, 23 nm) and by modifying the Kd of the bioreceptor using wild-type and mutant hDHFR. Furthermore, initial binding rate data analyzes demonstrated quantitative and fast sensor response under 60s. This MTX assay was subsequently adapted to a fully integrated multi-channel SPR system built in-house and calibrated in human serum with a dynamic range of 28-500 nM. The SPR system was applied to analyzes of actual clinical samples and the results are in good agreement with fluorescence polarization immunoassay (FPIA) and LC-MS/MS. Finally, the prototype system was tested by potential clinical users in a hospital setting at the biochemistry laboratory of a Montreal hospital (Hôpital Maisonneuve-Rosemont).

Non-specific adsorption of crude cell lysate on surface plasmon resonance sensors.

Non-specific adsorption of the molecular components of biofluids is ubiquitous in the area of biosensing technologies, severely limiting the use of biosensors in real-world applications. The surface chemistries developed to prevent non-specific adsorption of crude serum are not necessarily suited for sensing in other biosamples. In particular, the diagnostic potential of differential expression of proteins in tissues makes cell lysate attractive for disease diagnostics using solid biopsies. However, crude cell lysate poses a significant challenge for surface chemistries because of a large concentration of highly adherent lipids. Contrary to the non-specific adsorption in crude serum being suppressed by hydrophilic surfaces, the surface plasmon resonance (SPR) analysis of serine-, aspartic-acid-, histidine-, leucine-, and phenylalanine-based peptide monolayers revealed that hydrophobic and positively charged peptides decreased non-specific adsorption when using lysate from HEK 293FT cells. A polyethylene glycol (PEG) monolayer resulted in 2-fold greater fouling than the best peptide [3-MPA-(His)2(Leu)2(Phe)2-OH] under the same conditions. Matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analysis of the adsorbate from cell lysate confirmed that lipids are the main source of non-specific adsorption. Importantly, the mass spectrometry (MS) study revealed that both the number of lipids identified and their intensity decreased with decreasing non-specific adsorption. A peptide monolayer thus provides an efficient mean to suppress non-specific adsorption from this human cell lysate.

Modern surface plasmon resonance for bioanalytics and biophysics.

Physical chemistry, materials science, analytical chemistry and engineering greatly contributed to the increasing popularity of bioanalytical and biophysical applications of surface plasmon resonance (SPR) by providing novel materials, surface chemistry, instrumental concepts, and theory to further understand the plasmonic phenomenon and support innovation in SPR. This perspective article portrays the contemporary state of SPR-based techniques and establishes a list of challenges to be overcome for improving bioanalytical and biophysical applications of plasmonics and surface plasmon resonance.

Monitoring methotrexate in clinical samples from cancer patients during chemotherapy with a LSPR-based competitive sensor.

A competitive binding assay based on localized surface plasmon resonance (LSPR) of folic acid-functionalized gold nanoparticles (FA-AuNPs) and human dihydrofolate reductase enzyme (hDHFR) was developed to detect nanomolar to micromolar concentrations of the widely applied anti-cancer drug, methotrexate (MTX). By the nature of the competitive assay for MTX, the LSPR shift from specific binding between FA-AuNPs and the free enzyme was inversely proportional to the concentration of MTX. In addition, the dynamic range for MTX was tuned from 10(-11) to 10(-6) M by varying the concentration of hDHFR from 1 to 100 nM. Inter-day reproducibility and recovery of MTX spiked in phosphate buffer saline (PBS) were excellent. Potential interferents such as FA, trimethoprim (TMP) and 4-amino-4-deoxy-N-methylpteroic acid (DAMPA) did not occur in the concentration range of interest for MTX. Clinical samples of human serum from patients undergoing MTX chemotherapy were analyzed following a simple solid-phase extraction step to isolate MTX from the serum matrix, with a limit of detection of 155 nM. Validation of the LSPR method was carried out in comparison to Fluorescence Polarization Immunoassay (FPIA), a commonly used method in clinical settings, and LC-MS/MS, a reference technique. The results of the LSPR competitive assay compared well to FPIA and LC-MS/MS, with a slope of 2.4 and 1.1, respectively, for the correlation plots. The method established herein is intended for therapeutic drug monitoring (TDM) of MTX levels in patients undergoing chemotherapy to ensure safety and efficacy of the treatment.