Machine learning assisted mid-infrared spectrochemical fibrillar collagen imaging in clinical tissues.

Significance: Label-free multimodal imaging methods that can provide complementary structural and chemical information from the same sample are critical for comprehensive tissue analyses. These methods are specifically needed to study the complex tumor-microenvironment where fibrillar collagen's architectural changes are associated with cancer progression. To address this need, we present a multimodal computational imaging method where mid-infrared spectral imaging (MIRSI) is employed with second harmonic generation (SHG) microscopy to identify fibrillar collagen in biological tissues. Aim: To demonstrate a multimodal approach where a morphology-specific contrast mechanism guides a mid-infrared spectral imaging method to detect fibrillar collagen based on its chemical signatures. Approach: We trained a supervised machine learning (ML) model using SHG images as ground truth collagen labels to classify fibrillar collagen in biological tissues based on their mid-infrared hyperspectral images. Five human pancreatic tissue samples (sizes are in the order of millimeters) were imaged by both MIRSI and SHG microscopes. In total, 2.8 million MIRSI spectra were used to train a random forest (RF) model. The remaining 68 million spectra were used to validate the collagen images generated by the RF-MIRSI model in terms of collagen segmentation, orientation, and alignment. Results: Compared to the SHG ground truth, the generated MIRSI collagen images achieved a high average boundary F-score (0.8 at 4 pixels threshold) in the collagen distribution, high correlation (Pearson's R 0.82) in the collagen orientation, and similarly high correlation (Pearson's R 0.66) in the collagen alignment. Conclusions: We showed the potential of ML-aided label-free mid-infrared hyperspectral imaging for collagen fiber and tumor microenvironment analysis in tumor pathology samples.

Metasurface-Enhanced Mid-Infrared Spectrochemical Imaging of Tissues.

Label-free and nondestructive mid-infrared vibrational hyperspectral imaging is an essential tissue analysis tool, providing spatially resolved biochemical information critical to understanding physiological and pathological processes. However, the chemically complex and spatially heterogeneous composition of tissue specimens and the inherently weak interaction of infrared light with biomolecules limit the analytical performance of infrared absorption spectroscopy. Here, an advanced mid-infrared spectrochemical tissue imaging modality is introduced using metasurfaces that support strong surface-localized electromagnetic fields to capture quantitative molecular maps of large-area murine brain tissue sections. The approach leverages polarization-multiplexed multi-resonance plasmonic metasurfaces to simultaneously detect various functional biomolecules. The surface-enhanced mid-infrared spectral imaging method eliminates the non-specific effects of bulk tissue morphology on quantitative spectral analysis and improves chemical selectivity. This study shows that metasurface enhancement increases the retrieval of amide I and II bands associated with protein secondary structures. Moreover, it is demonstrated that plasmonic metasurfaces enhance the chemical contrast in infrared images and enable detection of ultrathin tissue regions that are not otherwise visible to conventional mid-infrared spectral imaging. While this work uses murine brain tissue sections, the chemical imaging method is well-suited for other tissue types, which broadens its potential impact for translational research and clinical histopathology.

Multiplexed COVID-19 antibody quantification from human sera using label-free nanoplasmonic biosensors.

Serological assays that can reveal immune status against COVID-19 play a critical role in informing individual and public healthcare decisions. Currently, antibody tests are performed in central clinical laboratories, limiting broad access to diverse populations. Here we report a multiplexed and label-free nanoplasmonic biosensor that can be deployed for point-of-care antibody profiling. Our optical imaging-based approach can simultaneously quantify antigen-specific antibody response against SARS-CoV-2 spike and nucleocapsid proteins from 50 µL of human sera. To enhance the dynamic range, we employed multivariate data processing and multi-color imaging and achieved a quantification range of 0.1-100 µg/mL. We measured sera from a COVID-19 acute and convalescent (N = 24) patient cohort and negative controls (N = 5) and showed highly sensitive and specific past-infection diagnosis. Our results were benchmarked against an electrochemiluminescence assay and showed good concordance (R∼0.87). Our integrated nanoplasmonic biosensor has the potential to be used in epidemiological sero-profiling and vaccine studies.

Imaging-based spectrometer-less optofluidic biosensors based on dielectric metasurfaces for detecting extracellular vesicles.

Biosensors are indispensable tools for public, global, and personalized healthcare as they provide tests that can be used from early disease detection and treatment monitoring to preventing pandemics. We introduce single-wavelength imaging biosensors capable of reconstructing spectral shift information induced by biomarkers dynamically using an advanced data processing technique based on an optimal linear estimator. Our method achieves superior sensitivity without wavelength scanning or spectroscopy instruments. We engineered diatomic dielectric metasurfaces supporting bound states in the continuum that allows high-quality resonances with accessible near-fields by in-plane symmetry breaking. The large-area metasurface chips are configured as microarrays and integrated with microfluidics on an imaging platform for real-time detection of breast cancer extracellular vesicles encompassing exosomes. The optofluidic system has high sensing performance with nearly 70 1/RIU figure-of-merit enabling detection of on average 0.41 nanoparticle/µm2 and real-time measurements of extracellular vesicles binding from down to 204 femtomolar solutions. Our biosensors provide the robustness of spectrometric approaches while substituting complex instrumentation with a single-wavelength light source and a complementary-metal-oxide-semiconductor camera, paving the way toward miniaturized devices for point-of-care diagnostics.

Rapid and Digital Detection of Inflammatory Biomarkers Enabled by a Novel Portable Nanoplasmonic Imager.

New point-of-care diagnostic devices are urgently needed for rapid and accurate diagnosis, particularly in the management of life-threatening infections and sepsis, where immediate treatment is key. Sepsis is a critical condition caused by systemic response to infection, with chances of survival drastically decreasing every hour. A novel portable biosensor based on nanoparticle-enhanced digital plasmonic imaging is reported for rapid and sensitive detection of two sepsis-related inflammatory biomarkers, procalcitonin (PCT) and C-reactive protein (CRP) directly from blood serum. The device achieves outstanding limit of detection of 21.3 pg mL-1 for PCT and 36 pg mL-1 for CRP, and dynamic range of at least three orders of magnitude. The portable device is deployed at Vall d'Hebron University Hospital in Spain and tested with a wide range of patient samples with sepsis, noninfectious systemic inflammatory response syndrome (SIRS), and healthy subjects. The results are validated against ultimate clinical diagnosis and currently used immunoassays, and show that the device provides accurate and robust performance equivalent to gold-standard laboratory tests. Importantly, the plasmonic imager can enable identification of PCT levels typical of sepsis and SIRS patients in less than 15 min. The compact and low-cost device is a promising solution for assisting rapid and accurate on-site sepsis diagnosis.

Optical Interrogation Techniques for Nanophotonic Biochemical Sensors.

The manipulation of light via nanoengineered surfaces has excited the optical community in the past few decades. Among the many applications enabled by nanophotonic devices, sensing has stood out due to their capability of identifying miniscule refractive index changes. In particular, when free-space propagating light effectively couples into subwavelength volumes created by nanostructures, the strongly-localized near-fields can enhance light's interaction with matter at the nanoscale. As a result, nanophotonic sensors can non-destructively detect chemical species in real-time without the need of exogenous labels. The impact of such nanophotonic devices on biochemical sensor development became evident as the ever-growing research efforts in the field started addressing many critical needs in biomedical sciences, such as low-cost analytical platforms, simple quantitative bioassays, time-resolved sensing, rapid and multiplexed detection, single-molecule analytics, among others. In this review, the optical transduction methods used to interrogate optical resonances of nanophotonic sensors will be highlighted. Specifically, the optical methodologies used thus far will be evaluated based on their capability of addressing key requirements of the future sensor technologies, including miniaturization, multiplexing, spatial and temporal resolution, cost and sensitivity.

Early sepsis diagnosis via protein and miRNA biomarkers using a novel point-of-care photonic biosensor.

Sepsis is a condition characterized by a severe stage of blood-infection often leading to tissue damage, organ failure and finally death. Fast diagnosis and identification of the sepsis stage (sepsis, severe sepsis or septic shock) is critical for the patient's evolution and could help in defining the most adequate treatment in order to reduce its mortality. The combined detection of several biomarkers in a timely, specific and simultaneous way could ensure a more accurate diagnosis. We have designed a new optical point-of-care (POC) device based on a phase-sensitive interferometric biosensor with a label-free microarray configuration for potential high-throughput evaluation of specific sepsis biomarkers. The sensor chip, which relies on the use of metallic nanostructures, provides versatility in terms of biofunctionalization, allowing the efficient immobilization of different kind of receptors such as antibodies or oligonucleotides. We have focused on two structurally different types of biomarkers: proteins, including C-reactive protein (CRP) and Interleukin 6 (IL6), and miRNAs, using miRNA-16 as an example. Limits of Detection (LoD) of 18 µg mL-1, 88 µg mL-1 and 1 µM (6 µg mL-1) have been respectively obtained for CRP, IL6 and miRNA-16 in individual assays, with high accuracy and reproducibility. The multiplexing capabilities have also been assessed with the simultaneous analysis of both protein biomarkers.

Publisher Correction: Self-assembly of nanostructured glass metasurfaces via templated fluid instabilities.

In the version of this Article originally published, the volume, article number and year of ref. 32 were incorrect; they should have read 31, 1802348 (2019). This has now been corrected.

Self-assembly of nanostructured glass metasurfaces via templated fluid instabilities.

Modern devices require the tuning of the size, shape and spatial arrangement of nano-objects and their assemblies with nanometre-scale precision, over large-area and sometimes soft substrates. Such stringent requirements are beyond the reach of conventional lithographic techniques or self-assembly approaches. Here, we show nanoscale control over the fluid instabilities of optical thin glass films for the fabrication of self-assembled all-dielectric optical metasurfaces. We show and model the tailoring of the position, shape and size of nano-objects with feature sizes below 100 nm and with interparticle distances down to 10 nm. This approach can generate optical nanostructures over rigid and soft substrates that are more than tens of centimetres in size, with optical performance and resolution on a par with advanced traditional lithography-based processes. To underline the potential of our approach, which reconciles high-performance optical metasurfaces and simple self-assembly fabrication approaches, we demonstrate experimentally and via numerical simulation sharp Fano resonances with a quality factor, Q, as high as ∼300 in the visible for all-dielectric nanostructures, to realize protein monolayer detection.

Label-free Bacteria Quantification in Blood Plasma by a Bioprinted Microarray Based Interferometric Point-of-Care Device.

Existing clinical methods for bacteria detection lack speed, sensitivity, and, importantly, point-of-care (PoC) applicability. Thus, finding ways to push the sensitivity of clinical PoC biosensing technologies is crucial. Here we report a portable PoC device based on lens-free interferometric microscopy (LIM). The device employs high performance nanoplasmonics and custom bioprinted microarrays and is capable of direct label-free bacteria ( E. coli) quantification. With only one-step sample handling we offer a sample-to-data turnaround time of 40 min. Our technology features detection sensitivity of a single bacterial cell both in buffer and in diluted blood plasma and is intrinsically limited by the number of cells present in the detection volume. When employed in a hospital setting, the device has enabled accurate categorization of sepsis patients (infectious SIRS) from control groups (healthy individuals and noninfectious SIRS patients) without false positives/negatives. User-friendly on-site bacterial clinical diagnosis can thus become a reality.

Imaging-based molecular barcoding with pixelated dielectric metasurfaces.

Metasurfaces provide opportunities for wavefront control, flat optics, and subwavelength light focusing. We developed an imaging-based nanophotonic method for detecting mid-infrared molecular fingerprints and implemented it for the chemical identification and compositional analysis of surface-bound analytes. Our technique features a two-dimensional pixelated dielectric metasurface with a range of ultrasharp resonances, each tuned to a discrete frequency; this enables molecular absorption signatures to be read out at multiple spectral points, and the resulting information is then translated into a barcode-like spatial absorption map for imaging. The signatures of biological, polymer, and pesticide molecules can be detected with high sensitivity, covering applications such as biosensing and environmental monitoring. Our chemically specific technique can resolve absorption fingerprints without the need for spectrometry, frequency scanning, or moving mechanical parts, thereby paving the way toward sensitive and versatile miniaturized mid-infrared spectroscopy devices.

Nanoparticle-Enhanced Plasmonic Biosensor for Digital Biomarker Detection in a Microarray.

Nanoplasmonic devices have become a paradigm for biomolecular detection enabled by enhanced light-matter interactions in the fields from biological and pharmaceutical research to medical diagnostics and global health. In this work, we present a bright-field imaging plasmonic biosensor that allows visualization of single subwavelength gold nanoparticles (NPs) on large-area gold nanohole arrays (Au-NHAs). The sensor generates image heatmaps that reveal the locations of single NPs as high-contrast spikes, enabling the detection of individual NP-labeled molecules. We implemented the proposed method in a sandwich immunoassay for the detection of biotinylated bovine serum albumin (bBSA) and human C-reactive protein (CRP), a clinical biomarker of acute inflammatory diseases. Our method can detect 10 pg/mL of bBSA and 27 pg/mL CRP in 2 h, which is at least 4 orders of magnitude lower than the clinically relevant concentrations. Our sensitive and rapid detection approach paired with the robust large-area plasmonic sensor chips, which are fabricated using scalable and low-cost manufacturing, provides a powerful platform for multiplexed biomarker detection in various settings.

Phase-sensitive plasmonic biosensor using a portable and large field-of-view interferometric microarray imager.

Nanophotonics, and more specifically plasmonics, provides a rich toolbox for biomolecular sensing, since the engineered metasurfaces can enhance light-matter interactions to unprecedented levels. So far, biosensing associated with high-quality factor plasmonic resonances has almost exclusively relied on detection of spectral shifts and their associated intensity changes. However, the phase response of the plasmonic resonances have rarely been exploited, mainly because this requires a more sophisticated optical arrangement. Here we present a new phase-sensitive platform for high-throughput and label-free biosensing enhanced by plasmonics. It employs specifically designed Au nanohole arrays and a large field-of-view interferometric lens-free imaging reader operating in a collinear optical path configuration. This unique combination allows the detection of atomically thin (angstrom-level) topographical features over large areas, enabling simultaneous reading of thousands of microarray elements. As the plasmonic chips are fabricated using scalable techniques and the imaging reader is built with low-cost off-the-shelf consumer electronic and optical components, the proposed platform is ideal for point-of-care ultrasensitive biomarker detection from small sample volumes. Our research opens new horizons for on-site disease diagnostics and remote health monitoring.

Plasmonic nanohole array biosensor for label-free and real-time analysis of live cell secretion.

Cell secretion dynamics plays a central role in physiological and disease processes. Due to its various temporal profiles, it is essential to implement a precise detection scheme for continuous monitoring of secretion in real time. The current fluorescent and colorimetric approaches hinder such applications due to their multiple time-consuming steps, molecular labeling, and especially the 'snapshot' endpoint readouts. Here, we develop a nanoplasmonic biosensor for real-time monitoring of live cell cytokine secretion in a label-free configuration. Our nanoplasmonic biosensor is composed of gold nanohole arrays supporting extraordinary optical transmission (EOT), which enables sensitive and high-throughput analysis of biomolecules. The nanobiosensor is integrated with an adjustable microfluidic cell module for the analysis of live cells under well-controlled culture conditions. We achieved an outstanding sensitivity for the detection of vascular endothelial growth factor (VEGF) directly in complex cell media. Significantly, the secretion dynamics from live cancer cells were monitored and quantified for 10 hours while preserving good cell viability. This novel approach of probing cytokine secretion activity is compatible with conventional inverted microscopes found in a common biology laboratory. With its simple optical set-up and label-free detection configuration, we anticipate our nanoplasmonic biosensor to be a powerful tool as a lab-on-chip device to analyze cellular activities for fundamental cell research and biotechnologies.