Label-free Optical Detection Research Articles

Gold nanoparticles assisted colorimetric sensing of paroxetine, duloxetine, and olanzapine in aqueous and micellar systems with improved sensitivity

The present study explored a rapid and facile colorimetric identification of two antidepressants (paroxetine and duloxetine) and one antipsychotic drug (olanzapine) in aqueous and micellar systems using citrate-stabilized gold nanoparticles (Cit-AuNPs). The sensing approach involved hydrogen bonding and electrostatic interactions on the surface of Cit-AuNPs. The attributes of Cit-AuNPs and their interactions with the drugs were probed using characterization techniques like UV–Vis spectroscopy, scanning electron microscopy (SEM), dynamic light scattering (DLS), zeta potential measurement, and Fourier-transform infrared spectroscopy (FT-IR). Various factors including the volume of Cit-AuNPs, pH, reaction time, surfactant concentration, and the sequence of addition of reagents were optimized to inspect their impacts on sensing results. Under optimized conditions, the probe showed good linearity for the three drugs at the nanomolar (nM) range. Consequently, the color of the Cit-AuNP solution transformed from red to royal purple and blue, which was visible to the naked eye and monitored by visible absorption spectra. This label-free optical detection approach could recognize paroxetine (Prx), duloxetine (Dlx), and olanzapine (Olz) in aqueous system at concentrations ranging from 1 to 17 nM with a limit of detection (LOD) of 0.312, 0.258, and 0.418 nM (S/N = 3), respectively. Meanwhile, the sensitivity was improved in the Triton X-100 (TX-100) micellar system with LOD of 0.029, 0.030, and 0.029 nM for Prx, Dlx, and Olz, respectively accompanied by a shift in the absorption bands. The micellar system contributes stability to the Cit-AuNP-drug complex. The devised colorimetric approach was convenient and did not require any complex equipment. It was efficaciously used to detect three drugs in pharmaceutical products, spiked human urine, and blood serum samples with satisfactory recoveries of 99.83–103.45% and RSD < 3.5%. Notably, the sensing assay demonstrated good selectivity for three drugs in the existence of commonly encountered foreign species and certain common drugs with a relative error below 3%. The results of the present study were statistically compared with reference methodology. The results were validated with no significant difference between the devised and reported approaches considering precision and accuracy, respectively.

Plasmonic Imaging of Single DNA with Charge Sensitive Monolayer WS2.

Imaging the surface charge of biomolecules such as proteins and DNA, is crucial for comprehending their structure and function. Unfortunately, current methods for label-free, sensitive, and rapid imaging of the surface charge of single DNA molecules are limited. Here, we propose a plasmonic microscopy strategy that utilizes charge-sensitive single-crystal monolayer WS2 materials to image the local charge density of a single λ-DNA molecule. Our study reveals that WS2 is a highly sensitive charge-sensitive material that can accurately measure the local charge density of λ-DNA with high spatial resolution and sensitivity. The consistency of the surface charge density values obtained from the single-crystal monolayer WS2 materials with theoretical simulations demonstrates the reliability of our approach. Our findings suggest that this class of materials has significant implications for the development of label-free, scanning-free, and rapid optical detection and charge imaging of biomolecules.

Label-Free and Real-Time Optical Detection of Affinity Binding of the Antibody on Adherent Live Cells.

Oblique-incidence reflectivity difference (OIRD) is a novel real-time, label-free, and nondestructive optical detection method and exhibits encouraging application in the detection of antibody/DNA microarrays. In this study, for the first time, an OIRD label-free immunoassay was achieved by using adherent live cells as the probe. The cells were cultured on glass cells, and the affinity binding of antibodies targeted on the HLA class I antigen of the cell surface was detected with an OIRD. The results show that an OIRD is able to detect the binding process of anti-human HLA-A, B, and C antibodies on MDA-MB-231 cells and HUVEC cells. Control experiments and complementary fluorescence analysis confirmed the high detection specificity and good quantitative virtue of the OIRD label-free immunoassay. Label-free OIRD imaging analysis of cell microarrays was further demonstrated successfully, and the underlying optical mechanism was revealed by combining the theoretical modeling. This work explores the use of live cells as probes for an OIRD immunoassay, thus expanding the potential applications of the OIRD in the field of pathological analysis, disease diagnosis, and drug screening, among others.

Label-free liquid crystal-based optical detection of norfloxacin using an aptamer recognition probe in soil and lake water.

Norfloxacin (NOX), a broad spectrum fluoroquinolone (FQ) antibiotic, is commonly detected in environmental residues, potentially contributing to biological drug resistance. In this paper, an aptamer recognition probe has been used to develop a label-free liquid crystal-based biosensor for simple and robust optical detection of NOX in aqueous solutions. Stimuli-receptive liquid crystals (LCs) have been employed to report aptamer-target binding events at the LC-aqueous interface. The homeotropic alignment of LCs at the aqueous-LC interface is due to the self-assembly of the cationic surfactant cetyltrimethylammonium bromide (CTAB). In the presence of the negatively charged NOX aptamer, the ordering changes to planar/tilted. On addition of NOX, the aptamer-NOX binding causes redistribution of CTAB at the LC-aqueous interface and the homeotropic orientation is restored. This results in a bright-to-dark optical transition under a polarized optical microscope (POM). This optical transition serves as a visual indicator to mark the presence of NOX. The devised aptasensor demonstrates high specificity with a minimum detection limit of 5 nM (1.596 ppb). Moreover, the application of the developed aptasensor for the detection of NOX in freshwater and soil samples underscores its practical utility in environmental monitoring. This proposed LC-based method offers several advantages over conventional detection techniques for a rapid, feasible and convenient way to detect norfloxacin.

Label-Free Identification of Biochemical Footprints of Key Mutations in Acute Myeloid Leukemia Using Raman Spectroscopy

Introduction: With increasing understanding of the complexity and heterogeneity of acute myeloid leukemia (AML), the diagnostic process becomes more complicated and time-consuming. Therefore, there is a need to supplement AML diagnostics with a quick method to identify key, druggable genetic lesions that serve as a target for small molecule drugs. To address this need, we used Raman spectroscopy (RS)-based imaging methods. RS uses non-elastic scattering of laser light on cell biomolecules. When coupled to computational image segmentation and analysis, composition of the analyzed fragment of the cytoplasm can be characterized. In this way, by mapping the focal surface of the cell, we obtain a multidimensional image containing information about the distribution of its biochemical components. In our study, we utilized RS microscopy to investigate how single mutations affect the Raman spectral images of leukemic cells. The primary objective was to determine whether this technique could be utilized to identify mutations in key genes related to leukemia development. Methods: Genetic models of AML were generated by introducing wild-type and mutated forms of three frequently mutated and/or druggable genes (FLT3, IDH1/2, and MLL) into myeloid cell lines (THP1, HEL, HL60). The presence of the desired phenotype was confirmed using flow cytometry, immunoblotting, high-throughput kinase activity assay (Pamgene), and confocal microscopy. For RS experiments, the cells were fixed, and subjected to Raman imaging measurements using the WITec Alpha 300 microscope. Reference or control samples consisted of cells with wild-type genes (WT). The recorded hyperspectral data was subjected to multidimensional chemometric analysis, including k-means cluster analysis (KMCA), principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). To validate the findings from the cell models, primary AML cells from 30 patients diagnosed with FLT-3 ITD, MLL or IDH1/2 mutations were examined with RS. Peripheral blood mononuclear cells (PBMC) isolated from healthy donors blood served as reference. Results: The spectral differences between cells with wild-type FLT3 and FLT3/ITD were subtle, yet significant. The results revealed distinct variances in lipid (1440, 1240, 1660, and 2850 cm -1) and hemoprotein content (740-760 cm -1). Additionally, we successfully distinguished cells harboring IDH1/2 and MLL mutations from those with the wild-type genes. In these cases Raman bands corresponding to protein and nucleic acids composition were changed in comparison to control cells. Raman features in PCA loadings were also related to proteins and nucleic acids. Using the PLS-DA method and simple AI model, we built successful algorithm for distinguishing transgenic models carrying mutation from wild-type cells. PCA analysis also enabled us to distinguish AML primary cells from PBMC. Analysis weather primary cells carrying the selected mutations can be distinguished from non-mutated AML cells is concordant with our findings on models, however, it requires further analysis in larger group of patients to increase statistical power of these observations. Conclusions: Collectively, these findings suggest that Raman spectroscopy coupled with appropriate chemometric analysis may serve as rapid, label-free tool for identification of key druggable mutations in AML. Acknowledgements: The studies were performed as a part of the “ Label-free and rapid optical imaging, detection and sorting of leukemia cells” project carried out within the Team-Net programme of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund.

Raman Spectroscopy Imaging in Evaluation of Immune Cells Activation Status

Introduction: The ability to assess the activation status of immune cells is useful for medical diagnostics and currently also for monitoring the performance of immune cells-based advanced therapy medicinal products (ATMP) such as the chimeric antigen receptor (CAR)-T cells. CAR-T cells numbers and immunophenotype are routinely monitored to avoid poor expansion or exhaustion phenotype. In this study, we utilized Raman spectroscopy (RS) as a label-free, non-destructive method for assessing T cells activation and macrophages polarization (pro-inflammatory M1 vs immunosuppressive M2). RS is a well-defined, non-elastic light scattering-based spectroscopic technique that followed by chemometric analysis provides unique and comprehensive information regarding the molecular structure, metabolic signature and biochemical composition of tissues and cells at subcellular level. To assess if RS would be a valid approach for detection of CAR-T activation we first developed CD19CAR-transduced Jurkat cell line model. Although Jurkat cells, a leukemic T cell line, lack cytolytic activity, they do produce IL-2 and upregulate CD69 upon activation like CAR-T thus serve as a convenient model. Materials and Methods: T cells and monocyte were isolated from healthy donors' blood. T cells were activated with coated CD3/CD28 beads. Jurkat cells were transduced with anti-CD19 chimeric TCR. CAR-J cells activation upon contact with CD19+ cells were confirmed by CD69 expression (flow cytometry) and calcium fluxes (fluorescence microscopy). Monocyte were differentiated to macrophages using CSF1, while THP1 monocytic cell line with PMA. Macrophages were subsequently polarized into M1 and M2 with IL-4 and IL-13 or IFN-gamma and LPS respectively. M0 macrophages or naïve T cells were used as a reference and control. Cell phenotypes were confirmed using flow cytometry, q-PCR, fluorescent confocal microscopy and proliferation assay. Cells were fixed in 0.5% glutaraldehyde and imaged at 533 and 633 nm with a WITec Alpha 300 Raman microscope. Collected data were analyzed by chemometric methods: K-means cluster analysis (KMCA) followed by Partial Least Squares-Discrimination Analysis (PLS-DA) and Principal Component Analysis (PCA). Results: RS analysis of polarized macrophages showed the increased signal from hemoproteins (740-760 cm -1) in all three states. Interestingly we observed different hemoproteins to lipids (1440, 1240, 1660, 2850 cm -1) ratio between M0, M1 and M2. Also degree of lipid unsaturation can be considered an important parameter in the identification of the M1 phenotype of macrophages. Chemometric analyzes revealed significant morphological (increased cell and nucleus sizes) biochemical and difference between activated T lymphocytes and naive cells. Carotenoids present in non-activated T cells disappeared after activation. Conversely, lipids in the form of distinct clusters were observed after activation. Moreover, in the spectra of cell nuclei (733, 790, 1583 cm -1) of activated lymphocytes, less condensed chromatin was manifested by a lower intensity of marker bands. We observed similar changes for activated CAR-Jurkat, though the morphological changes were more subtle and carotenoids were not observed in Jurkat cells. Conclusions: The obtained results indicate the potential utility of Raman spectroscopy methods in identifying lymphocyte states activation and macrophage polarization . Acknowledgements. The studies were performed as a part of the “ Label-free and rapid optical imaging, detection and sorting of leukemia cells” project carried out within the Team-Net programme of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund.

Raman Spectroscopy Imaging As a Tool to Classify Diffused Large B-Cell Lymphoma Subtypes

INTRODUCTION: Diffuse large B-cell lymphoma (DLBCL) is a clinically heterogenous, aggressive disease. The ‘cell-of-origin’ classification (COO) distinguishes two major DLBCL subtypes, GCB (germinal center B cell-like) and ABC (activated B cell-like) that are derived from distinct putative cell of origin, differ in prognosis and transcriptional pattern. Comprehensive cluster classification (CCC) based on unbiased “consensus clustering” of transcriptomic microarray data has led to the identification of subtypes that differ in energy source utilization and bioenergetic profiles The oxPhos DLBCL subtype, unlike non-oxPhos, is characterized by increased mitochondrial oxidative phosphorylation and energy transduction. Although COO class assignment can be achieved with multiple clinical diagnostic tools (including transcript-based or immunohistochemical approaches), there is no clinical diagnostic tool appropriate for COO classification. In Raman effect, laser light, as a result of internal oscillations of molecules, is scattered with a change in wavelength, giving a spectrum characteristic for a given substance. This allows the chemical composition of mixtures and even cells to be determined on the basis of the obtained spectra. Thus, it also allows to determine differences in metabolism, including energy metabolism of lymphoma cells. The aim of the study was to use the RS images to recognize and classify DLBCL cells. MATERIALS AND METHODS: DLBCL cell lines (OCI-Ly4, Pfeiffer, Toledo, DHL4, DHL6 , OCI-Ly1 and OCI-Ly7) with known COO and CCC subtype assignments, and B cells isolated from the blood of healthy donors were used. For Raman analysis cells were fixed in 0.5% glutaraldehyde and imaged at 533 and 633 nm emission wavelengths under a WITec Raman microscope. The collected spectral images were subjected to chemometric analyses: characteristic bands analysis, k-means cluster analysis (KMCA), principal component analysis (PCA) and least squares discriminant analysis (PLS-DA) using dedicated software. RESULTS: The analysis of Raman spectra revealed clear changes in the structure and composition of cells after neoplastic transformation. Chemometric analysis of spectral bands showed greater proportion of proteins (1008, 1041 and 1660 cm-1) and lipids (1440, 1240 and 2850 cm-1) in DLBCL cells then B cells from healthy donors. More nucleic acids were observed (733, 790 , 1583 cm-1) in normal B cells that at least partially resulted from the greater ratio of the nucleus to the cytoplasm. The differences resulted in clear separation of malignant and healthy cells in PCA and PLS-DA. Similar approach allowed to characterize differences between ABC vs GCB subtype cell lines and oxPhos vs non-oxPhos subtype. Based on the specific differences between healthy B cells and DLBCL cells a predictive model was built using PLS-DA that accurately determines the chance of the analyzed cell, first, being a lymphoma cell, and second, belonging to the ABC vs GCB or oxPhos vs non-oxPhos DLBCL subtype. The good performance of the constructed predictive model was evaluated by prediction on unknown samples with sensitivity and specificity of 98%. CONCLUSIONS: We present a model of a classification of B cell lymphomas based on label-free Raman spectral imagines, capable of automatic and efficient identification of DLBCL cells spectra. In addition, we describe each lymphoma subtype by its unique spectral profile, linking it to biochemical features. Acknowledgements. The studies were performed as a part of the “ Label-free and rapid optical imaging, detection and sorting of leukemia cells” project carried out within the Team-Net programme of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund.

Ultrasensitive, dynamic, and online monitoring photonic sensors for protein conformation

The monitoring of molecular dynamic transition and protein conformation changes is important for some neurodegenerative diseases (NDs) and has attracted much attention due to its ability to reveal the pathological mechanism of several NDs. Here, a label-free optical detection method with an ultrahigh refractive index sensitivity (S) and dynamical S tuning range was developed to provide a novel technology roadmap for the online monitoring of protein conformation. The real-time conformation variation process was transduced into a visualization optical signal, allowing it to be monitored. The biosensor prototype was constructed by the cross-overlapping of two rows of square capillaries on a gold mirror to form two microcavities with different free spectral ranges (denoted as the sensing and reference cavities). The coupling of the resonance mode between the two microcavities induced the generation of the Vernier effect and the magnifying of S, presented in the form of a modulated envelope in the spectrum. S values ranged from −17,256 nm/RIU to 93,937 nm/RIU, indicating that the biosensor could not only realize large dynamic tuning ranges but also an ultrahigh S. The conformation changes of silk proteins from the random coil to the β–sheet were sensitively monitored by the output spectrum after it was stimulated with ethanol solution. This ultrasensitive biosensor (which is both label-free and optical) provides a novel and supplementary technique to monitor the structural and conformation changes process of proteins. The results reveal its significant potential for clinical diagnosis, healthcare, pharmaceutical screening, and food security.

Vapor-Phase Synthesis of Molecularly Imprinted Polymers on Nanostructured Materials at Room-Temperature.

Molecularly imprinted polymers (MIPs) have recently emerged as robust and versatile artificial receptors. MIP synthesis is carried out in liquid phase and optimized on planar surfaces. Application of MIPs to nanostructured materials is challenging due to diffusion-limited transport of monomers within the nanomaterial recesses, especially when the aspect ratio is >10. Here, the room temperature vapor-phase synthesis of MIPs in nanostructured materials is reported. The vapor phase synthesis leverages a >1000-fold increase in the diffusion coefficient of monomers in vapor phase, compared to liquid phase, to relax diffusion-limited transport and enablethe controlled synthesis of MIPs also in nanostructures with high aspect ratio. As proof-of-concept application, pyrrole is used as the functional monomer thanks to its large exploitation in MIP preparation; nanostructured porous silicon oxide (PSiO2 ) is chosen to assess the vapor-phase deposition of PPy-based MIP in nanostructures with aspect ratio >100; human hemoglobin (HHb) is selected as the target molecule for the preparation of a MIP-based PSiO2 optical sensor. High sensitivityand selectivity, low detection limit, high stability and reusability are achieved in label-free optical detection of HHb, also in human plasma and artificial serum. The proposed vapor-phase synthesis of MIPs is immediately transferable to other nanomaterials, transducers, and proteins.

Label-free optical detection and quantification of pathogens for point-of-care applications

Label-free optical detection and quantification of pathogens for point-of-care applications

Design and Characterization of ITO-Covered Resonant Nanopillars for Dual Optical and Electrochemical Sensing

In this work we present a dual optical and electrochemical sensor based on SiO2/Si3N4 resonant nanopillars covered with an indium tin oxide (ITO) thin film. A 25–30 nm thick ITO layer deposited by magnetron sputtering acts as an electrode when incorporated onto the nanostructured array, without compromising the optical sensing capability of the nanopillars. Bulk sensing performances before and after ITO deposition have been measured and compared in accordance with theoretical calculations. The electrochemical activity has been determined by the ferri/ferrocyanide redox reaction, showing a remarkably higher activity than that of flat thin films of similar ITO nominal thickness, and proving that the nanopillar system covered by ITO presents electrical continuity. A label-free optical biological detection has been performed, where the presence of amyloid-β has been detected through an immunoassay enhanced with gold nanoparticles. Again, the experimental results have been corroborated by theoretical simulations. We have demonstrated that ITO can be a beneficial component for resonant nanopillars sensors by adding potential electrochemical sensing capabilities, without significantly altering their optical properties. We foresee that resonant nanopillars coated with a continuous ITO film could be used for simultaneous optical and electrochemical biosensing, improving the robustness of biomolecular identification.

(Electronics and Photonics Division Award) Let There be Light; Research at the Intersection of Electronics and Photonics

Electronics and photonics have a long and successful history of combining to provide many devices and products that are at the heart of the modern technological world, ranging form solar cells to lasers to digital displays. In this presentation I will first give a brief historical overview of the interplay and interdependence of electronics and photonics. The properties and effects that are commonly exploited in opto/electronics fall primarily in two areas: (a) the effect of incident light (or more generally radiation) due to optical absorption/transmission/reflection and the conversion of photons to electrical charge that can be collected; (b) the injection of electrons & holes leading to the emission of photons. Then I will review selected examples from my research in optoelectronic materials and devices in more or less chronological order over the past 40+ years. An early example [1] in the light-to-charge was integration of infrared detectors using narrow bandgap semiconductors (such as InSb and HgCdTe) with silicon charge coupled devices that performed data storage and image processing functions on arrays of pixels. A major research topic in my group in the charge-to-light category has for many years been electroluminescence [2] from wide bandgap semiconductors (primarily GaN and AlN) doped with rare earth elements (Er, Eu, Tm, etc.). Narrow linewidth emission can occur at wavelengths from near UV to visible to IR, depending on the electronic transitions of the selected RE element and the bandgap of the host material. In the past 10 years, my materials interest switched from inorganic to organic semiconductors and biomaterials, but with a continuing strong opto/electronic component. Currently, a major research focus is in developing simple, low cost and intuitive diagnostic devices for point-of-care use. These devices analyze the composition of biofluids (e.g. blood, sweat, saliva, etc.) to provide a 1st order indication of specific aspects the individual’s health status, such as blood coagulation, stress, etc. The use of light in PoC and other diagnostic devices is widespread. Most common and simplest approach is colorimetric detection in ambient light, where a change of color in a region of the device indicates the presence of a specific biomolecule associated with a given medical condition. Somewhat more complex diagnostic devices use fluorescence of molecules attached to biomarkers to indicate their presence. In our own research, biofluid samples are typically transported by capillary flow to a location where their key properties are measured by a combination of optical emission [3], transmission and absorption [4]. The presentation will conclude with a look forward to other applications where photonics and electronics could join forces.[1] A.J. Steckl, et al., “Application of Charge Coupled Devices to Infrared Detection and Imaging,” Proc. of IEEE, 63(1), 67, Jan 1975 ( doi.org/10.1109/PROC.1975.9708 ).[2] (a) A.J. Steckl and J.M. Zavada, “Optoelectronic Properties and Applications of Rare-Earth-Doped GaN”, Mat. Res. Soc. Bulletin, 24(9), 33, Sep 1999 doi.org/10.1557/S0883769400053045; (b) A. J. Steckl, et al., Invited Paper, “Rare-earth-doped GaN: Growth, Properties and Fabrication of Electroluminescent Devices”, IEEE J. Selected Topics in Quantum Electronics, 8 (4), 749 Jul 2002 doi.org/10.1109/JSTQE.2002.801690 [3] V. Venkatraman and A. J. Steckl, “Quantitative Detection in Lateral Flow Immunoassay Using Integrated Organic Optoelectronics”, IEEE Sensors, 17 (24), 8343, Dec 2017 (doi.org/10.1109/JSEN.2017.2764178 ).[4] P. Ray and A. J. Steckl, “Label-Free Optical Detection of Multiple Stress Biomarkers in Sweat, Plasma, Urine and Saliva”, ACS Sensors 4, 1346, 2019. (doi.org/10.1021/acssensors.9b00301). Figure 1

Label-Free Optical Detection of Pathogenic Bacteria and Fungi at Extremely Low Cell Densities for Rapid Antibiotic Susceptibility Testing.

Antibiotic resistance is a rapidly expanding public health problem across the globe leading to prolonged hospital admissions, increased morbidity and mortality, and associated high healthcare costs. Effective treatment of bacterial infections requires timely and correct antibiotic administration to the patients which relies on rapid phenotyping of disease-causing bacteria. Currently, antibiotic susceptibility tests can take several days and as a result, indiscriminate antibiotic use has exacerbated the evolution and spread of antibiotic resistance in clinical and community settings. In order to address this problem, we have developed a novel optical apparatus that we called RUSD (Rapid Ultra-Sensitive Detection). RUSD is built around a hollow silica fiber and utilizes bacterial cells as spatial light modulators. This generates a highly sensitive modulation transfer function due to the narrow reflectivity angle in the fiber-media interface. We leveraged the RUSD technology to allow for robust bacterial and fungal detection. RUSD can now detect pathogenic cell densities in a large dynamic window (OD600 from ∼10−7 to 10−1). Finally, we can generate dose response curves for various pathogens and antimicrobial compounds within one to three hours by using RUSD. Our antibiotic- susceptibility testing (AST) assay that we call iFAST (in-Fiber-Antibiotic-Susceptibility-Testing) is fast, highly sensitive, and does not change the existing workflow in clinical settings as it is compatible with FDA-approved AST. Thus, RUSD platform is a viable tool that will expedite decision-making process in the treatment of infectious diseases and positively impact the antibiotic resistance problem in the long term by minimizing the use of ineffective antibiotics.

Docking and Activity of DNA Polymerase on Solid-State Nanopores.

Integration of motor enzymes with biological nanopores has enabled commercial DNA sequencing technology; yet studies of the similar principle applying to solid-state nanopores are limited. Here, we demonstrate the real-life monitoring of phi29 DNA polymerase (DNAP) docking onto truncated-pyramidal nanopore (TPP) arrays through both electrical and optical readout. To achieve effective docking, atomic layer deposition of hafnium oxide is employed to reduce the narrowest pore opening size of original silicon (Si) TPPs to sub-10 nm. On a single TPP with pore opening size comparable to DNAP, ionic current measurements show that a polymerase–DNA complex can temporally dock onto the TPP with a certain docking orientation, while the majority become translocation events. On 5-by-5 TPP arrays, a label-free optical detection method using Ca2+ sensitive dye, are employed to detect the docking dynamics of DNAP. The results show that this label-free detection strategy is capable of accessing the docking events of DNAP on TPP arrays. Finally, we examine the activity of docked DNAP by performing on-site rolling circle amplification to synthesize single-stranded DNA (ssDNA), which serves as a proof-of-concept demonstration of utilizing this docking scheme for emerging nanopore sensing applications.

Self-assembled 1D nanostructures for direct nanoscale detection and biosensing

•Self-assembled 1D nanostructures allow for nanoscale detection and visualization •Enabled direct measurement of nanoscale objects at the single-particle level •Suitable for rapid and ultrasensitive sensing of virus samples, including SARS-CoV-2 Directly accurate detection of nanoscale objects remains a great challenge to many fields, including self-assembly, nanoparticle engineering, nanomaterial characterization, and biological diagnosis. Here, we report a general method for visualizable detection and measurement of many nanoscale objects by using one-dimensional (1D) nanostructures, including nanoparticles, defects, and pathogens. The large-scale 1D nanostructures are robustly prepared through the template-assisted self-assembly of colloidal nanoparticles, which exhibit wide spectral tunability in the visible region. In addition to the scalability, they can significantly amplify the weak optical signals from nanoscale objects, pushing the detection sensitivity down to the single-particle level. Then, we achieve the rapid and ultrasensitive testing of different clinical virus samples by using a simple optical microscope or a portable cell phone. This work demonstrates a label-free, simple, and high-efficiency optical detection platform at the nanoscale, which has a broad range of applications from material synthesis to in vitro diagnosis. Directly accurate detection of nanoscale objects remains a great challenge to many fields, including self-assembly, nanoparticle engineering, nanomaterial characterization, and biological diagnosis. Here, we report a general method for visualizable detection and measurement of many nanoscale objects by using one-dimensional (1D) nanostructures, including nanoparticles, defects, and pathogens. The large-scale 1D nanostructures are robustly prepared through the template-assisted self-assembly of colloidal nanoparticles, which exhibit wide spectral tunability in the visible region. In addition to the scalability, they can significantly amplify the weak optical signals from nanoscale objects, pushing the detection sensitivity down to the single-particle level. Then, we achieve the rapid and ultrasensitive testing of different clinical virus samples by using a simple optical microscope or a portable cell phone. This work demonstrates a label-free, simple, and high-efficiency optical detection platform at the nanoscale, which has a broad range of applications from material synthesis to in vitro diagnosis.

A Compact Surface Plasmon Resonance Biosensor for Sensitive Detection of Exosomal Proteins for Cancer Diagnosis.

Exosomes are nanosized (50-150nm) extracellular vesicles released by all types of cells in the body. They transport various biological molecules, such as DNAs, RNAs, proteins, and lipids from parent cells to recipient cells for intercellular communication. Exosomes, especially those from tumor cells, are actively involved in caner development, metastasis, and drug resistance. Recently, many studies have shown that exosomal proteins are promising biomarkers for cancer screening, early detection and prognosis. Among many detection techniques, surface plasmon resonance (SPR) is a highly sensitive, label-free, and real-time optical detection method. Commercial prism-based wavelength/angular-modulated SPR sensors afford high sensitivity and resolution, but their large footprint and high cost limit their adaptability for clinical settings. We have developed an intensity-modulated, compact SPR biosensor (25cm×10cm×25cm) for the detection of exosomal proteins. We have demonstrated the potential application of the compact SPR biosensor in lung cancer diagnosis using exosomal epidermal growth factor receptor (EGFR) and programmed death-ligand 1 (PD-L1) as biomarkers. The compact SPR biosensor offers sensitive, simple, fast, user-friendly, and cost-effective detection of exosomal proteins, which may serve as an in vitro diagnostic test for cancer.

Charge Sensitive Optical Detection for Measurement of Small-Molecule Binding Kinetics.

Charge sensitive optical detection (CSOD) technique is a label-free method for real-time measurement of molecular interactions. Traditional label-free optical detection techniques mostly measure the mass of a molecule, and they are less sensitive to small molecules. In contrast, CSOD detects the charge of a molecule, where the signal does not diminish with the size of the molecule, thus capable for studying small molecules. In addition, CSOD is compatible with the standard microplate platform, making it suitable for high-throughput screening of drug candidates. In CSOD, an optical fiber functionalized with the probe molecule is dipped into a well of a microplate where an alternate perpendicular electrical field is applied to the fiber, which drives the fiber into oscillation because of the presence of surface charge on the fiber. The binding of the target molecules changes the charge of the fiber, and thus the amplitude and phase of the oscillating fiber, which are precisely measured through tracking of the optical images of the fiber tip.

Printed Nanochain-Based Colorimetric Assay for Quantitative Virus Detection.

Fast and ultrasensitive detection of pathogens is very important for efficient monitoring and prevention of viral infections. Here, we demonstrate a label-free optical detection approach that uses a printed nanochain assay for colorimetric quantitative testing of viruses. The antibody-modified nanochains have high activity and specificity which can rapidly identify target viruses directly from biofluids in 15 min, as well as differentiate their subtypes. Arising from the resonance induced near-field enhancement, the color of nanochains changes with the binding of viruses that are easily observed by a smartphone. We achieve the detection limit of 1 PFU μL-1 through optimizing the optical response of nanochains in visible region. Besides, it allows for real-time response to virus concentrations ranging from 0 to 1.0×105 PFU mL-1 . This low-cost and portable platform is also applicable to rapid detection of other biomarkers, making it attractive for many clinical applications.

Electrically Controlled Nanophotonic Slot Structure Based on Photocatalytic Nanocomposite for Optical Detection of Foodborne Pathogens

A nanophotonic structure with electric control based photocatalytic nanocomposite is proposed to realize label-free optical detection of foodborne pathogens. The photocatalytic response of Cu-ZnO/TiO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> (CZT) nanocomposite, in terms of the photocurrent, is utilized to detect E. coli in the surrounding media of Luria Bertani broth (LB) solution. An engineered nanophotonic structure based on an optical slot waveguide is presented to provide a two-fold benefit - enhanced light-matter interaction and flexibility to accommodate bio-sample. CZT nanocomposite is synthesised and prepared by a cost-effective sol-gel process, acting as an active layer in the structure, is responsible for the generation of photo-excited carriers which provide photocurrent on the application of a voltage. The fabricated structure with a synthesised nanocomposite slot waveguide exhibits a measured photocurrent of 22 μA with LB media, decreasing to a value of 13 μA in the presence of E. coli. The fabricated biosensor is capable of detecting E. coli bacteria concentrations of 5000 CFU/ml. Altogether a change in photocurrent of 9 μA is obtained with E. coli on applying a low voltage of 3.5 volts. The combination of photocatalytic nanocomposite with engineered optical waveguide carries great potential for applications in biochemical detection and in realizing other integrated photonic devices with slotted nanocomposites.

Detecting attomolar concentrations of microRNA related to myelodysplastic syndromes in blood plasma using a novel sandwich assay with nanoparticle release

Microribonucleic acids (miRNAs) are short noncoding ribonucleic acids that have been linked with a multitude of human diseases including lung, breast, and hematological cancers. In this work, we present a novel, extremely sensitive assay for the label-free optical biosensor-based detection of miRNAs, which is based on the oligonucleotide-triggered release of nanoparticles from a sensor surface. We combine this assay (herein referred to as the nanoparticle-release (NPR) assay) with a surface plasmon resonance biosensor and show that the assay is able to enhance the specific sensor response associated with the binding of target miRNA while suppressing the interfering effects caused by the non-specific binding. We apply the assay to the detection of miRNAs related to myelodysplastic syndromes (miR-125b, miR-16) in blood plasma and demonstrate that the assay enables detection of miR-125b with a limit of detection (LOD) of 349 aM (corresponding to the lowest detectable amounts of 419 zmol). The achieved LOD is better by a factor of ∼100 when compared to the conventional nanoparticle-enhanced sandwich assay. Moreover, we demonstrate that the NPR assay may be combined with time-division multiplexing for the multiplexed miRNA detection.