Plasmonic Functional Assay Platform Determines the Therapeutic Profile of Cancer Cells.

Functional assay platforms could identify the biophysical properties of cells and their therapeutic response to drug treatments. Despite their strong ability to assess cellular pathways, functional assays require large tissue samples, long-term cell culture, and bulk measurements. Even though such a drawback is still valid, these limitations did not hinder the interest in these platforms for their capacity to reveal drug susceptibility. Some of the limitations could be overcome with single-cell functional assays by identifying subpopulations using small sample volumes. Along this direction, in this article, we developed a high-throughput plasmonic functional assay platform to identify the growth profile of cells and their therapeutic profile under therapies using mass and growth rate statistics of individual cells. Our technology could determine populations' growth profiles using the growth rate data of multiple single cells of the same population. Evaluating spectral variations based on the plasmonic diffraction field intensity images in real time, we could simultaneously monitor the mass change for the cells within the field of view of a camera with the capacity of > ∼500 cells/h scanning rate. Our technology could determine the therapeutic profile of cells under cancer drugs within few hours, while the classical techniques require days to show reduction in viability due to antitumor effects. The platform could reveal the heterogeneity within the therapeutic profile of populations and determine subpopulations showing resistance to drug therapies. As a proof-of-principle demonstration, we studied the growth profile of MCF-7 cells and their therapeutic behavior to standard-of-care drugs that have antitumor effects as shown in the literature, including difluoromethylornithine (DFMO), 5-fluorouracil (5-FU), paclitaxel (PTX), and doxorubicin (Dox). We successfully demonstrated the resistant behavior of an MCF-7 variant that could survive in the presence of DFMO. More importantly, we could precisely identify synergic and antagonistic effects of drug combinations based on the order of use in cancer therapy. Rapidly assessing the therapeutic profile of cancer cells, our plasmonic functional assay platform could be used to reveal personalized drug therapies for cancer patients.

An optofluidic platform for cell-counting applications.

Cell-counting is critical for a wide range of applications, e.g., life sciences, medicine, or pharmacology. Hemocytometry is a classical method that requires manual counting of cells under a microscope. This methodology is low-cost but manual counting is slow, and the test accuracy is limited by the operator experience. Accuracy and throughput of such application could be improved with the use of automated cell-counting devices. Possessing the ability of recording and processing cell images, devices employing these technologies could dramatically improve the accuracy of the counting results. However, accuracy of these devices still requires further improvement as the counting results rely only on 100-200 cells. Furthermore, the test cost of these devices increases due to the need for a counting chamber or consumables compatible with their hardware settings. Herein, in order to address these drawbacks, we introduced an optofluidic cell-counting platform that could scan more than 2000 cells, which dramatically improves the test accuracy. Our technology could yield an error rate below 1% for cell viability, and below 5% for cell concentration. The platform could deliver the count results within only ∼1 minute, including sample loading, autofocusing, recording images, and image processing. The presented platform also benefits from a built-in fluidic component that eliminates the need for an external counting chamber, and allows fully automated sample loading and self-cleaning modality compatible with any solutions that are typically used for cell-counting tests. Providing an easy-to-use and rapid feature from sample loading to image analyses, our optofluidic platform could be a critical asset for accurate and low cost cell-counting applications.

Interaction of nickel ferrite nanoparticles with nucleic acids.

In this article, we introduced an electrochemical biosensor employing graphite electrodes (GE) decorated with Nickel ferrite (NiFe2O4) nanoparticles for nucleic acid detection. NiFe2O4 nanoparticles in a narrow size distribution were synthesized with co-precipitation technique. Their chemical and crystallographic properties were characterized with FTIR and X-ray spectroscopies. Nanoparticle size distribution and hydrodynamic diameter were determined with particle size analyzer. Elemental content and purity of nanoparticles were analyzed with EDX analysis. Our analyses showed a diameter of ~10 nm for NiFe2O4 nanoparticles. Electrochemical properties of NiFe2O4 nanoparticles were examined with different analysis methods. Conductivity properties of NiFe2O4 nanoparticles were investigated with Cyclic Voltammetry (CV), which confirmed that nanoparticles on GE surface have a high surface area and conductivity. More importantly, in this article, the interactions between NiFe2O4 nanoparticles and double stranded DNA (dsDNA), single stranded DNA (ssDNA), and RNA were for the first time examined using Differential Pulse Voltammetry (DPV), CV, and Electrochemical Impedance Spectroscopy (EIS). Oxidation peak currents of NiFe2O4 nanoparticles and guanine bases of dsDNA, ssDNA, and RNA showed that NiFe2O4 nanoparticles effectively interacts with nucleic acids via an electrostatic mode.

Electrochemical Detection of Linagliptin and its Interaction with DNA

Objectives: Linagliptin (Lin) is a drug used in treatment of type 2 diabetes mellitus. In this study, the electrochemical detection of Lin and its interaction with DNA was analyzed for the first time using voltammetric methods by measuring the oxidation currents of the adenine bases of DNA before and after the interaction. In addition, the electrochemical properties of the Lin were studied. Materials and Methods: The interaction between Lin and DNA was evaluated using differential pulse voltammetry. A three-electrode system comprising of a pencil graphite electrode as the working electrode, reference electrode (Ag/AgCl), and platinum wire as the auxiliary electrode was used in the electrochemical studies. Experimental conditions, such as the concentration, pH of the supporting electrolyte, and immobilization time were optimized to obtain maximum analytical signals. Results: The adenine bases of DNA were evaluated as an analytical signal obtained at approximately +1.2 V vs. Ag/AgCl. After the Lin-DNA interaction, the oxidation currents of adenine decreased as proof of interaction. No reports have been published on Lin interacting with DNA. Based on our results, a diffusion-controlled irreversible redox process involving independent oxidation was revealed for Lin. Under optimum conditions, the detection limit was 6.7 µg/mL for DNA and 21.5 µg/mL for Lin. Based on the observations, Lin has a toxic effect on DNA. Conclusion: We successfully demonstrated that Lin interacts with DNA, and its influence on DNA could play a vital role in the medical effect of the drug.

Handheld plasmonic biosensor for virus detection in field-settings.

After World Health Organization (WHO) announced COVID-19 outbreak a pandemic, we all again realized the importance of developing rapid diagnostic kits. In this article, we introduced a lightweight and field-portable biosensor employing a plasmonic chip based on nanohole arrays integrated to a lensfree-imaging framework for label-free detection of viruses in field-settings. The platform utilizes a CMOS (complementary metal-oxide-semiconductor) camera with high quantum efficiency in the spectral window of interest to monitor diffraction field patterns of nanohole arrays under the uniform illumination of an LED (light-emitting diode) source which is spectrally tuned to the plasmonic mode supported by the nanohole arrays. As an example for the applicability of our biosensor for virus detection, we could successfully demonstrate the label-free detection of H1N1 viruses, e.g., swine flu, with medically relevant concentrations. We also developed a low-cost and easy-to-use sample preparation kit to prepare the surface of the plasmonic chip for analyte binding, e.g., virus-antibody binding. In order to reveal a complete biosensor technology, we also developed a user friendly Python™ - based graphical user interface (GUI) that allows direct access to biosensor hardware, taking and processing diffraction field images, and provides virus information to the end-user. Employing highly sensitive nanohole arrays and lensfree-imaging framework, our platform could yield an LOD as low as 103 TCID50/mL. Providing accurate and rapid sensing information in a handheld platform, weighing only 70 g and 12 cm tall, without the need for bulky and expensive instrumentation, our biosensor could be a very strong candidate for diagnostic applications in resource-poor settings. As our detection scheme is based on the use of antibodies, it could quickly adapt to the detection of different viral diseases, e.g., COVID-19 or influenza, by simply coating the plasmonic chip surface with an antibody possessing affinity to the virus type of interest. Possessing this ability, our biosensor could be swiftly deployed to the field in need for rapid diagnosis, which may be an important asset to prevent the spread of diseases before turning into a pandemic by isolating patients from the population.

Refractive Index Sensing for Measuring Single Cell Growth.

Accessing cell growth on adhesive substrates is critical for identifying biophysical properties of cells and their therapeutic response to drug therapies. However, optical techniques have low sensitivity, and their reliability varies with cell type, whereas microfluidic technologies rely on cell suspension. In this paper, we introduced a plasmonic functional assay platform that can precisely measure cell weight and the dynamic change in real-time for adherent cells. Possessing this ability, our platform can determine growth rates of individual cells within only 10 min to map the growth profile of populations in short time intervals. The platform could successfully determine heterogeneity within the growth profile of populations and assess subpopulations exhibiting distinct growth profiles. As a proof of principle, we investigated the growth profile of MCF-7 cells and the effect of two intracellular metabolisms critical for their proliferation. We first investigated the negative effect of serum starvation on cell growth. We then studied ornithine decarboxylase (ODC) activity, a key enzyme which is involved in proliferation, and degraded under low osmolarity that inhibits cell growth. We successfully determined the significant distinction between growth profiles of MCF-7 cells and their ODC-overproducing variants that possess strong resistance to the negative effects of low osmolarity. We also demonstrated that an exogenous parameter, putrescine, could rescue cells from ODC inhibition under hypoosmotic conditions. In addition to the ability of accessing intracellular activities through ex vivo measurements, our platform could also determine therapeutic behaviors of cancer cells in response to drug treatments. Here, we investigated difluoromethylornithine (DFMO), which has antitumor effects on MCF-7 cells by inhibiting ODC activity. We successfully demonstrated the susceptibility of MCF-7 cells to such drug treatment, while its DFMO-resistant subpopulation could survive in the presence of this antigrowth agent. By rapidly determining cell growth kinetics in small samples, our plasmonic platform may be of broad use to basic research and clinical applications.

Pathogen detection with electrochemical biosensors: Advantages, challenges and future perspectives.

Detection of pathogens, e.g., bacteria and viruses, is still a big challenge in analytical medicine due to their vast number and variety. Developing strategies for rapid, inexpensive, specific, and sensitive detection of the pathogens using nanomaterials, integrating with microfluidics devices, amplification methods, or even combining these strategies have received significant attention. Especially, after the health-threatening COVID-19 outbreak, rapid and sensitive detection of pathogens became very critical. Detection of pathogens could be realized with electrochemical, optical, mass sensitive, or thermal methods. Among them, electrochemical methods are very promising by bringing different advantages, i.e., they exhibit more versatile detection schemes and real-time quantification as well as label-free measurements, which provides a broader application perspective. In this review, we discuss the recent advances for the detection of bacteria and viruses using electrochemical biosensors. Moreover, electrochemical biosensors for pathogen detection were broadly reviewed in terms of analyte, bio-recognition and transduction elements. Different fabrication techniques, detection principles, and applications of various pathogens with the electrochemical biosensors were also discussed.

Correction: Synthesis and characterization of nanoceria for electrochemical sensing applications.

[This corrects the article DOI: 10.1039/D1RA00637A.].

Synthesis and characterization of nanoceria for electrochemical sensing applications.

Nanoceria (cerium oxide nanoparticles: CeO2-NPs) has received significant attention due to its biocompatibility, good conductivity, and the ability to transfer oxygen. Nanoceria has been widely used to develop electrochemical sensors and biosensors as it could increase response time, sensitivity, and stability of the sensor. In this review, we discussed synthesis methods, and the recent applications employing CeO2-NPs for electrochemical detection of various analytes reported in the most recent four years.

Plasmon-Coupled Photocapacitor Neuromodulators.

Efficient transduction of optical energy to bioelectrical stimuli is an important goal for effective communication with biological systems. For that, plasmonics has a significant potential via boosting the light-matter interactions. However, plasmonics has been primarily used for heat-induced cell stimulation due to membrane capacitance change (i.e., optocapacitance). Instead, here, we demonstrate that plasmonic coupling to photocapacitor biointerfaces improves safe and efficacious neuromodulating displacement charges for an average of 185% in the entire visible spectrum while maintaining the faradic currents below 1%. Hot-electron injection dominantly leads the enhancement of displacement current in the blue spectral window, and the nanoantenna effect is mainly responsible for the improvement in the red spectral region. The plasmonic photocapacitor facilitates wireless modulation of single cells at three orders of magnitude below the maximum retinal intensity levels, corresponding to one of the most sensitive optoelectronic neural interfaces. This study introduces a new way of using plasmonics for safe and effective photostimulation of neurons and paves the way toward ultrasensitive plasmon-assisted neurostimulation devices.

Photonic crystal and plasmonic nanohole based label-free biodetection.

Photonic crystals and plasmonic nanohole arrays are the conventional substrates for label-free biodetection applications. In this article, we readdressed these systems in terms of their sensing capability and provided a broad picture for a selection mechanism of optimum parameters providing strong sensing signals. We first investigated the physical origin of the transmission resonances supported by the two systems, which is the core of the label-free sensing mechanism, relying on strong light-matter interactions. We conducted an extensive theoretical study on optical and sensing properties of the two systems, e.g., linewidth of the optical modes, refractive index sensitivity and figure-of-merit capacities. Our theoretical analyses provided a rule-of-thumb method for the selection of geometrical device parameters of the two systems. In order to experimentally investigate the sensing properties, we fabricated the two systems via a lift-off free fabrication method based on electron beam lithography, where the plasmonic nanohole arrays are realized by covering the phonic crystal surface with a thin metal. As an example, we demonstrated the sensing strength of two systems with identical dimensions by monitoring the spectral variations within their optical responses. We also performed label-free sensing experiments through detection of protein mono- and bilayers, where the geometrical parameters favor the plasmonic sensor system. Integrating a high-resolution optical read-out scheme with a multi-spectral data tracking technique, we achieved an experimentally minimum detectable protein concentration as low as 200 pg/mL for the plasmonic nanohole array and 1 ng/mL for the photonic crystal-based sensing platform.

Plasmonic Sensor Could Enable Label-Free DNA Sequencing.

We demonstrated a proof-of-principle concept of a label-free platform that enables nucleic acid sequencing by binding methodology. The system utilizes gold surfaces having high fidelity plasmonic nanohole arrays which are very sensitive to minute changes of local refractive indices. Our novel surface chemistry approach ensures accurate identification of correct bases at individual positions along a targeted DNA sequence on the gold surface. Binding of the correct base on the gold sensing surface triggers strong spectral variations within the nanohole optical response, which provides a high signal-to-noise ratio and accurate sequence data. Integrating our label-free sequencing platform with a lens-free imaging-based device, we reliably determined targeted DNA sequences by monitoring the changes within the plasmonic diffraction images. Consequently, this new label-free surface chemistry technique, integrated with plasmonic lens-free imaging platform, will enable monitoring multiple biomolecular binding events, which could initiate new avenues for high-throughput nucleic acid sequencing.

Nitration of tyrosine and its effect on DNA hybridization.

One major marker of nitrosative stress is the formation of 3-Nitrotyrosine (3-NT) from Tyrosine (Tyr) by adding a nitro group (-NO2) with nitrating agents. Nitration of Tyr often causes loss of protein activity and is linked with many diseases. In this article, we detect 3-NT and discriminate it from Tyr with Differential Pulse Voltammetry (DPV) as it is a very important biomarker. We first examined redox (oxidation/reduction) properties and stability of 3-NT in detail. Second, we provided the Tyr and 3-NT discrimination with DPV and compared with the chromatography. We then explored the interaction of 3-NT and DNA oligonucleotides. Our findings demonstrate that 3-NT can be used as a new electrochemical indicator, which is able to detect hybridization of probe (single stranded DNA-ssDNA) and hybrid (double stranded DNA-dsDNA) both via 3-NT reduction and guanine oxidation signal changes at the same time. The signal differences enabled us to distinguish ssDNA and dsDNA without using a label or a tag. Moreover, we achieved to detect hybridization of DNA by using the reduction signal of 3-NT obtained at -0.4V vs. Ag/AgCl. More importantly, we observed the changes of the reduction signals of 3-NT after the interaction of probe and hybrid sequences. We showed that 3-NT signal decreases more with hybrid than the probe. Our platform, for the first time, demonstrates the detection of hybridization both guanine oxidation and indicator reduction signal changes at the same time. Moreover, we, for the first time, demonstrated the interaction between 3-NT and DNA.

Determining therapeutic susceptibility in multiple myeloma by single-cell mass accumulation.

Multiple myeloma (MM) has benefited from significant advancements in treatment that have improved outcomes and reduced morbidity. However, the disease remains incurable and is characterized by high rates of drug resistance and relapse. Consequently, methods to select the most efficacious therapy are of great interest. Here we utilize a functional assay to assess the ex vivo drug sensitivity of single multiple myeloma cells based on measuring their mass accumulation rate (MAR). We show that MAR accurately and rapidly defines therapeutic susceptibility across human multiple myeloma cell lines to a gamut of standard-of-care therapies. Finally, we demonstrate that our MAR assay, without the need for extended culture ex vivo, correctly defines the response of nine patients to standard-of-care drugs according to their clinical diagnoses. This data highlights the MAR assay in both research and clinical applications as a promising tool for predicting therapeutic response using clinical samples.

Lensfree optofluidic plasmonic sensor for real-time and label-free monitoring of molecular binding events over a wide field-of-view.

We demonstrate a high-throughput biosensing device that utilizes microfluidics based plasmonic microarrays incorporated with dual-color on-chip imaging toward real-time and label-free monitoring of biomolecular interactions over a wide field-of-view of >20 mm(2). Weighing 40 grams with 8.8 cm in height, this biosensor utilizes an opto-electronic imager chip to record the diffraction patterns of plasmonic nanoapertures embedded within microfluidic channels, enabling real-time analyte exchange. This plasmonic chip is simultaneously illuminated by two different light-emitting-diodes that are spectrally located at the right and left sides of the plasmonic resonance mode, yielding two different diffraction patterns for each nanoaperture array. Refractive index changes of the medium surrounding the near-field of the nanostructures, e.g., due to molecular binding events, induce a frequency shift in the plasmonic modes of the nanoaperture array, causing a signal enhancement in one of the diffraction patterns while suppressing the other. Based on ratiometric analysis of these diffraction images acquired at the detector-array, we demonstrate the proof-of-concept of this biosensor by monitoring in real-time biomolecular interactions of protein A/G with immunoglobulin G (IgG) antibody. For high-throughput on-chip fabrication of these biosensors, we also introduce a deep ultra-violet lithography technique to simultaneously pattern thousands of plasmonic arrays in a cost-effective manner.

Three-dimensional crystalline and homogeneous metallic nanostructures using directed assembly of nanoparticles.

Directed assembly of nano building blocks offers a versatile route to the creation of complex nanostructures with unique properties. Bottom-up directed assembly of nanoparticles have been considered as one of the best approaches to fabricate such functional and novel nanostructures. However, there is a dearth of studies on making crystalline, solid, and homogeneous nanostructures. This requires a fundamental understanding of the forces driving the assembly of nanoparticles and precise control of these forces to enable the formation of desired nanostructures. Here, we demonstrate that colloidal nanoparticles can be assembled and simultaneously fused into 3-D solid nanostructures in a single step using externally applied electric field. By understanding the influence of various assembly parameters, we showed the fabrication of 3-D metallic materials with complex geometries such as nanopillars, nanoboxes, and nanorings with feature sizes as small as 25 nm in less than a minute. The fabricated gold nanopillars have a polycrystalline nature, have an electrical resistivity that is lower than or equivalent to electroplated gold, and support strong plasmonic resonances. We also demonstrate that the fabrication process is versatile, as fast as electroplating, and scalable to the millimeter scale. These results indicate that the presented approach will facilitate fabrication of novel 3-D nanomaterials (homogeneous or hybrid) in an aqueous solution at room temperature and pressure, while addressing many of the manufacturing challenges in semiconductor nanoelectronics and nanophotonics.

Actively transporting virus like analytes with optofluidics for rapid and ultrasensitive biodetection.

Effective analyte delivery is essential to achieve rapid and sensitive biodetection systems. In this article, we present an actively controlled fluidic system integrated with a suspended plasmonic nanohole sensor to achieve superior analyte delivery efficiency and ultrafast sensor response, as compared to conventional fluidic systems. 70 nm sized virus like analyte solution is used to experimentally demonstrate the system performance improvements. Sensor response time is reduced by one order of magnitude as compared to the conventional methods. A seven orders of magnitude dynamic concentration range from 10(3) to 10(9) particles mL(-1) is quantified, corresponding to a concentration window relevant to clinical diagnosis and drug screening. Our non-destructive detection system, by enabling efficient analyte delivery, fast sensing response and minimal sample volume, opens up opportunities for sensitive, rapid and real-time virus detection in infectious disease control and point-of-care applications.

Fano resonant ring/disk plasmonic nanocavities on conducting substrates for advanced biosensing.

By introducing a conducting metal layer underneath a Fano resonant asymmetric ring/disk plasmonic nanocavity system, we demonstrate that electromagnetic fields can be strongly enhanced. These large electromagnetic fields extending deep into the medium are highly accessible and increase the interaction volume of analytes and optical fields. As a result, we demonstrate high refractive index sensitivities as large as 648 nm/RIU. By exciting Fano resonances with much sharper spectral features, as narrow as 9 nm, we experimentally show high figure of merits as large as 72 and reliable detection of protein mono- and bilayers. Furthermore, the conducting substrate enables strong interaction between fundamental and higher order modes of the system by minor structural asymmetries. This is very advantageous for experimental realization of systems supporting resonances with well-defined Fano-like line shape without requiring challenging fabrication resolution. Exploiting conducting metallic substrates and the associated propagating surface plasmons at their interface could be extended to other Fano resonant cavity geometries for improved biosensing performance.

Plasmon induced transparency in cascaded π-shaped metamaterials.

We experimentally and numerically demonstrate a planar metamaterial consisting of two asymmetrically positioned π-structures in a single unit that exhibits plasmonic analogue of electromagnetically induced transparency (EIT). Through the coupling of the constituent nanorod elements, the proposed structure enables fine spectral tuning of the EIT-like behavior and controlling the location of near field enhancement. Originated from the asymmetric cascaded π-structures, we introduce a more compact system which possesses the EIT-like characteristics and as well as much smaller mode volumes. Due to these properties, the proposed metamaterials can be utilized for a wide range of applications including bio-chemical sensors, optical filters and modulators and enhancement of non-linear processes.

Seeing protein monolayers with naked eye through plasmonic Fano resonances.

We introduce an ultrasensitive label-free detection technique based on asymmetric Fano resonances in plasmonic nanoholes with far reaching implications for point-of-care diagnostics. By exploiting extraordinary light transmission phenomena through high-quality factor (Q(solution) ∼ 200) subradiant dark modes, we experimentally demonstrate record high figures of merits (FOMs as high as 162) for intrinsic detection limits surpassing that of the gold standard prism coupled surface-plasmon sensors (Kretschmann configuration). Our experimental record high sensitivities are attributed to the nearly complete suppression of the radiative losses that are made possible by the high structural quality of the fabricated devices as well as the subradiant nature of the resonances. Steep dispersion of the plasmonic Fano resonance profiles in high-quality plasmonic sensors exhibit dramatic light intensity changes to the slightest perturbations within their local environment. As a spectacular demonstration of the extraordinary sensitivity and the quality of the fabricated biosensors, we show direct detection of a single monolayer of biomolecules with naked eye using these Fano resonances and the associated Wood's anomalies. To fabricate high optical-quality sensors, we introduce a high-throughput lift-off free evaporation fabrication technique with extremely uniform and precisely controlled nanofeatures over large areas, leading to resonance line-widths comparable to that of the ideally uniform structures as confirmed by our time-domain simulations. The demonstrated label-free sensing platform offers unique opportunities for point-of-care diagnostics in resource poor settings by eliminating the need for fluorescent labeling and optical detection instrumentation (camera, spectrometer, etc.) as well as mechanical and light isolation.