An Automated and Fast Sample Preparation Workflow for Laser Microdissection Guided Ultrasensitive Proteomics.

Spatial tissue proteomics integrating whole-slide imaging, laser microdissection, and ultrasensitive mass spectrometry is a powerful approach to link cellular phenotypes to functional proteome states in (patho)physiology. To be applicable to large patient cohorts and low sample input amounts, including single-cell applications, loss-minimized and streamlined end-to-end workflows are key. We here introduce an automated sample preparation protocol for laser microdissected samples utilizing the cellenONE robotic system, which has the capacity to process 192 samples in 3 h. Following laser microdissection collection directly into the proteoCHIP LF 48 or EVO 96 chip, our optimized protocol facilitates lysis, formalin de-crosslinking, and tryptic digest of low-input archival tissue samples. The seamless integration with the Evosep ONE LC system by centrifugation allows 'on-the-fly' sample clean-up, particularly pertinent for laser microdissection workflows. We validate our method in human tonsil archival tissue, where we profile proteomes of spatially-defined B-cell, T-cell, and epithelial microregions of 4000 µm2 to a depth of ∼2000 proteins and with high cell type specificity. We finally provide detailed equipment templates and experimental guidelines for broad accessibility.

Ultrasensitive Protein Detection Technologies for Extracellular Vesicle Measurements.

Extracellular vesicles (EVs) are nanoscopic, heterogenous, lipid-rich particles that carry a multitude of cargo biomolecules including proteins, nucleic acids, and metabolites. Although historically EVs were regarded as cellular debris with no intrinsic value, growing understanding of EV biogenesis has led to the realization that EVs facilitate intercellular communication and are sources of liquid biomarkers. EVs can be isolated and analyzed from a wide variety of accessible biofluids for biomarker discovery and diagnostic applications. There is a diversity of EVs from different biological compartments (e.g., cells and tissues), and some of these EVs are present at extremely low concentrations. Consequently, a challenge in the field is to find appropriate markers that enable selective isolation of these rare EVs. Many conventional protein detection technologies have limited sensitivity to detect low abundance biomarkers in EVs, limiting their use in EV research. Advances in ultrasensitive detection technologies are needed to harness the potential of EVs for clinical application. This Perspective highlights current EV research focusing on ultrasensitive detection technologies, their limitations, and areas of potential growth in the future.

Dynamic single-molecule sensing by actively tuning binding kinetics for ultrasensitive biomarker detection.

SignificanceThe detection of low-abundance molecular biomarkers is key to the liquid-biopsy-based disease diagnosis. Existing methods are limited by the affinity and specificity of recognition probes and the mass transportation of analyte molecules onto the sensor surfaces, resulting in insufficient sensitivity and long assay time. This work establishes a rapid and ultrasensitive approach by actively tuning binding kinetics and accelerating the mass transportation via nanoparticle micromanipulations. This is significant because it permits extremely sensitive measurements within clinically acceptable assay time. It is incubation-free, washing-free, and compatible with low- and high-affinity probes.

Ultrasensitive Photothermal Switching with Resonant Silicon Metasurfaces at Visible Bands.

Dynamic access to quasi-bound states in the continuum (q-BICs) offers a highly desired platform for silicon-based active nanophotonic applications, while the prevailing tuning approaches by free carrier injections via an all-optical stimulus are yet limited to THz and infrared ranges and are less effective in visible bands. In this work, we present the realization of active manipulations on q-BICs for nanoscale optical switching in the visible by introducing a local index perturbation through a photothermal mechanism. The sharp q-BIC resonance exhibits an ultrasensitive susceptibility to the complex index perturbation, which can be flexibly fulfilled by optical heating of silicon. Consequently, a mild pump intensity of 1 MW/cm2 can yield a modification of the imaginary part of the refractive index of less than 0.05, which effectively suppresses the sharp q-BIC resonances and renders an active modulation depth of reflectance exceeding 80%. Our research might open up an enabling platform for ultrasensitive dynamic nanophotonic devices.

Quantum Gas-Enabled Direct Mapping of Active Current Density in Percolating Networks of Nanowires.

Electrically percolating nanowire networks are among the most promising candidates for next-generation transparent electrodes. Scientific interest in these materials stems from their intrinsic current distribution heterogeneity, leading to phenomena like percolating pathway rerouting and localized self-heating, which can cause irreversible damage. Without an experimental technique to resolve the current distribution and an underpinning nonlinear percolation model, one relies on empirical rules and safety factors to engineer materials. We introduce Bose-Einstein condensate microscopy to address the longstanding problem of imaging active current flow in 2D materials. We report on performance improvement of this technique whereby observation of dynamic redistribution of current pathways becomes feasible. We show how this, combined with existing thermal imaging methods, eliminates the need for assumptions between electrical and thermal properties. This will enable testing and modeling individual junction behavior and hot-spot formation. Investigating both reversible and irreversible mechanisms will contribute to improved performance and reliability of devices.

An Ultrasensitive Ti3C2Tx MXene-based Soft Contact Lens for Continuous and Nondestructive Intraocular Pressure Monitoring.

Wearable soft contact lens sensors for continuous and nondestructive intraocular pressure (IOP) monitoring are highly desired as glaucoma and postoperative myopia patients grow, especially as the eyestrain crowd increases. Herein, a smart closed-loop system is presented that combines a Ti3C2Tx MXene-based soft contact lens (MX-CLS) sensor, wireless data transmission units, display, and warning components to realize continuous and nondestructive IOP monitoring/real-time display. The fabricated MX-CLS device exhibits an extremely high sensitivity of 7.483 mV mmHg-1, good linearity on silicone eyeballs, excellent stability under long-term pressure-release measurement, sufficient transparency with 67.8% transmittance under visible illumination, and superior biocompatibility with no discomfort when putting the MX-CLS sensor onto the Rabbit eyes. After integrating with the wireless module, users can realize real-time monitoring and warning of IOP via smartphones, the demonstrated MX-CLS device together with the IOP monitoring/display system opens up promising platforms for Ti3C2Tx materials as the base for multifunctional contact lens-based sensors and continuous and nondestructive IOP measurement system.

A critical evaluation of ultrasensitive single-cell proteomics strategies.

Success of mass spectrometry characterization of the proteome of single cells allows us to gain a greater understanding than afforded by transcriptomics alone but requires clear understanding of the tradeoffs between analytical throughput and precision. Recent advances in mass spectrometry acquisition techniques, including updated instrumentation and sample preparation, have improved the quality of peptide signals obtained from single cell data. However, much of the proteome remains uncharacterized, and higher throughput techniques often come at the expense of reduced sensitivity and coverage, which diminish the ability to measure proteoform heterogeneity, including splice variants and post-translational modifications, in single cell data analysis. Here, we assess the growing body of ultrasensitive single-cell approaches and their tradeoffs as researchers try to balance throughput and precision in their experiments.

Development of surface-enhanced Raman scattering-sensing Method by combining novel Ag@Au core/shell nanoparticle-based SERS probe with hybridization chain reaction for high-sensitive detection of hepatitis C virus nucleic acid.

The ultrasensitive detection of hepatitis C virus (HCV) nucleic acid is crucial for the early diagnosis of hepatitis C. In this study, by combining Ag@Au core/shell nanoparticle (Ag@AuNP)-based surface-enhanced Raman scattering (SERS) tag with hybridization chain reaction (HCR), a novel SERS-sensing method was developed for the ultrasensitive detection of HCV nucleic acid. This SERS-sensing system comprised two different SERS tags, which were constructed by modifying Ag@AuNP with a Raman reporter molecule of 4-ethynylbezaldehyde, two different hairpin-structured HCR sequences (H1 or H2), and a detection plate prepared by immobilizing a capture DNA sequence onto the Ag@AuNP layer surface of the detection wells. When the target nucleic acid was present, the two SERS tags were captured on the surface of the Ag@AuNP-coated detection well to generate many "hot spots" through HCR, forming a strong SERS signal and realizing the ultrasensitive detection of the target HCV nucleic acid. The limit of detection of the SERS-sensing method for HCV nucleic acid was 0.47 fM, and the linear range was from 1 to 105 fM.

Scaling Up Single-Cell Proteomics.

Single-cell tandem MS has enabled analyzing hundreds of single cells per day and quantifying thousands of proteins across the cells. The broad dissemination of these capabilities can empower the dissection of pathophysiological mechanisms in heterogeneous tissues. Key requirements for achieving this goal include robust protocols performed on widely accessible hardware, robust quality controls, community standards, and automated data analysis pipelines that can pinpoint analytical problems and facilitate their timely resolution. Toward meeting these requirements, this perspective outlines both existing resources and outstanding opportunities, such as parallelization, for catalyzing the wide dissemination of quantitative single-cell proteomics analysis that can be scaled up to tens of thousands of single cells. Indeed, simultaneous parallelization of the analysis of peptides and single cells is a promising approach for multiplicative increase in the speed of performing deep and quantitative single-cell proteomics. The community is ready to begin a virtuous cycle of increased adoption fueling the development of more technology and resources for single-cell proteomics that in turn drive broader adoption, scientific discoveries, and clinical applications.

Ultrasensitive, Multiplexed Buoyant Sensor for Monitoring Cytokines in Biofluids.

Multiplexed quantification of low-abundance protein biomarkers in complex biofluids is important for biomedical research and clinical diagnostics. However, in situ sampling without perturbing biological systems remains challenging. In this work, we report a buoyant biosensor that enables in situ monitoring of protein analytes at attomolar concentrations with a 15 min temporal resolution. The buoyant biosensor implemented with fluorescent nanolabels enabled the ultrasensitive and multiplexed detection and quantification of cytokines. Implementing the biosensor in a digital manner (i.e., counting the individual nanolabels) further improves the low detection limit. We demonstrate that the biosensor enables the detection and quantification of the time-varying concentrations of cytokines (e.g., IL-6 and TNF-α) in macrophage culture media without perturbing the live cells. The easy-to-apply biosensor with attomolar sensitivity and multiplexing capability can enable an in situ analysis of protein biomarkers in various biofluids and tissues to aid in understanding biological processes and diagnosing and treating diverse diseases.

A Self-Sustaining Near-Infrared Afterglow Chemiluminophore for High-Contrast Activatable Imaging.

Afterglow imaging holds great promise for ultrasensitive bioimaging due to its elimination of autofluorescence. Self-sustaining afterglow molecules (SAMs), which enable all-in-one photon sensitization, chemical defect formation and afterglow generation, possess a simplified, reproducible, and efficient superiority over commonly used multi-component systems. However, there is a lack of SAMs, particularly those with much brighter near-infrared (NIR) emission and structural flexibility for building high-contrast activatable imaging probes. To address these issues, this study for the first time reports a methylene blue derivative-based self-sustaining afterglow agent (SAN-M) with brighter NIR afterglow chemiluminescence peaking at 710 nm. By leveraging the structural flexibility and tunability, an activatable nanoprobe (SAN-MO) is customized for simultaneously activatable fluoro-photoacoustic and afterglow imaging of peroxynitrite (ONOO- ), notably with a superior activation ratio of 4523 in the afterglow mode, which is at least an order of magnitude higher than other reported activatable afterglow systems. By virtue of the elimination of autofluorescence and ultrahigh activation contrast, SAN-MO enables early monitoring of the LPS-induced acute inflammatory response within 30 min upon LPS stimulation and precise image-guided resection of tiny metastatic tumors, which is unattainable for fluorescence imaging.

Magneto-Nanomechanical Array Biosensor for Ultrasensitive Detection of Oncogenic Exosomes for Early Diagnosis of Cancers.

Exosomes are a class of nanoscale vesicles secreted by cells, which contain abundant information closely related to parental cells. The ultrasensitive detection of cancer-derived exosomes is highly significant for early non-invasive diagnosis of cancer. Here, an ultrasensitive nanomechanical sensor is reported, which uses a magnetic-driven microcantilever array to selectively detect oncogenic exosomes. A magnetic force, which can produce a far greater deflection of microcantilever than that produced by the intermolecular interaction force even with very low concentrations of target substances, is introduced. This method reduced the detection limit to less than 10 exosomes mL-1 . Direct detection of exosomes in the serum of patients with breast cancer and in healthy people showed a significant difference. This work improved the sensitivity by five orders of magnitude as compared to that of traditional nanomechanical sensing based on surface stress mode. This method can be applied parallelly for highly sensitive detection of other microorganisms (such as bacteria and viruses) by using different probe molecules, which can provide a supersensitive detection approach for cancer diagnosis, food safety, and SARS-CoV-2 infection.

At-Home COVID-19 Rapid Antigen Test Down to 0.03 pg mL-1 of Nucleocapsid Protein.

Rapid and ultra-sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for early screening and management of COVID-19. Currently, the real-time reverse transcription polymerase chain reaction (rRT-PCR) is the primary laboratory method for diagnosing SARS-CoV-2. It is not suitable for at-home COVID-19 diagnostic test due to the long operating time, specific equipment, and professional procedures. Here an all-printed photonic crystal (PC) microarray with portable device for at-home COVID-19 rapid antigen test is reported. The fluorescence-enhanced effect of PC amplifies the fluorescence intensity of the labeled probe, achieving detection of nucleocapsid (N-) protein down to 0.03 pg mL-1 . A portable fluorescence intensity measurement instrument gives the result (negative or positive) by the color of the indicator within 5 s after inserting the reacted PC microarray test card. The N protein in inactivated virus samples (with cycle threshold values of 26.6-40.0) can be detected. The PC microarray provides a general and easy-to-use method for the timely monitoring and eventual control of the global coronavirus pandemic.

Ultrasensitive Terahertz Biodetection Enabled by Quasi-BIC-Based Metasensors.

Advanced sensing devices, highly sensitive, and reliable in detecting ultralow concentrations of circulating biomarkers, are extremely desirable and hold great promise for early diagnostics and real-time progression monitoring of diseases. Nowadays, the most commonly used clinical methods for diagnosing biomarkers suffer from complicated procedures and being time consumption. Here, a chip-based portable ultra-sensitive THz metasensor is reported by exploring quasi-bound states in the continuum (quasi-BICs) and demonstrate its capability for sensing low-concentration analytes. The designed metasensor is made of the designed split-ring resonator metasurface which supports magnetic dipole quasi-BIC combining functionalized gold nanoparticles (AuNPs) conjugated with the specific antibody. Attributed to the strong near-field enhancement near the surface of the microstructure enabled by the quasi-BICs, light-analyte interactions are greatly enhanced, and thus the device's sensitivity is boosted significantly. The system sensitivity slope is up to 674 GHz/RIU, allowing for repeatable resolving detecting ultralow concentration of C-reactive protein (CRP) and Serum Amyloid A (SAA), respectively, down to 1 pM. The results touch a range that cannot be achieved by ordinary immunological assays alone, offering a novel non-destructive and rapid trace measured approach for next-generation biomedical quantitative detection systems.

A Novel Electrocatalyst Pd(II)@Ni3 (HITP)2 for Ultrasensitive Detection of Chloramphenicol: Experimental and Computational Investigation.

Ultrasensitive electrochemical detection on hazardous substances like antibiotics and pesticides is essential but still challenging in rapid test technology. Herein, the first electrode using highly conductive metal-organic frameworks (HCMOFs) for electrochemical detection of chloramphenicol is proposed. The design of electrocatalyst Pd(II)@Ni3 (HITP)2 with ultra-sensitivity in detection of chloramphenicol is demonstrated by loading Pd onto HCMOFs. An ultra-low limit of detection (LOD) of 0.2 nM (64.6 pg/mL) was observed for these materials, which is 1-2 orders of magnitude lower than the other reported materials for chromatographic detection. Moreover, the proposed HCMOFs showed long-time stability over 24 h. The superior detection sensitivity can be attributed to the high conductivity of Ni3 (HITP)2 , and the large Pd loading. The experimental characterizations and computational investigation determined the Pd loading mechanism in Pd(II)@Ni3 (HITP)2 , revealing PdCl2 adsorption on the abundant adsorption sites of Ni3 (HITP)2 . The proposed electrochemical sensor design using HCMOFs was demonstrated to be both effective and efficient, showing that the adoption of HCMOFs decorated with other effective electrocatalysts, which combine high conductivity and high catalytic activity, is of great advantage for ultrasensitive detection.

Biosensors, Recent Advances in Determination of BDNF and NfL.

Key biomarkers such as Brain Derived Neurotrophic Factor (BDNF) and Neurofilament light chain (NfL) play important roles in the development and progression of many neurological diseases, including multiple sclerosis, Alzheimer's disease, and Parkinson's disease. In these clinical conditions, the underlying biomarker processes are markedly heterogeneous. In this context, robust biomarker discovery is of critical importance for screening, early detection, and monitoring of neurological diseases. The difficulty of directly identifying biochemical processes in the central nervous system (CNS) is challenging. In recent years, biomarkers of CNS inflammatory response have been identified in various body fluids such as blood, cerebrospinal fluid, and tears. Furthermore, biotechnology and nanotechnology have facilitated the development of biosensor platforms capable of real-time detection of multiple biomarkers in clinically relevant samples. Biosensing technology is approaching maturity and will be deployed in communities, at which point screening programs and personalized medicine will become a reality. In this multidisciplinary review, our goal is to highlight clinical and current technological advances in the development of multiplex-based solutions for effective diagnosis and monitoring of neuroinflammatory and neurodegenerative diseases. The trend in the detection if BDNF and NfL.

Phenotypic heterogeneity in human genetic diseases: ultrasensitivity-mediated threshold effects as a unifying molecular mechanism.

Phenotypic heterogeneity is very common in genetic systems and in human diseases and has important consequences for disease diagnosis and treatment. In addition to the many genetic and non-genetic (e.g., epigenetic, environmental) factors reported to account for part of the heterogeneity, we stress the importance of stochastic fluctuation and regulatory network topology in contributing to phenotypic heterogeneity. We argue that a threshold effect is a unifying principle to explain the phenomenon; that ultrasensitivity is the molecular mechanism for this threshold effect; and discuss the three conditions for phenotypic heterogeneity to occur. We suggest that threshold effects occur not only at the cellular level, but also at the organ level. We stress the importance of context-dependence and its relationship to pleiotropy and edgetic mutations. Based on this model, we provide practical strategies to study human genetic diseases. By understanding the network mechanism for ultrasensitivity and identifying the critical factor, we may manipulate the weak spot to gently nudge the system from an ultrasensitive state to a stable non-disease state. Our analysis provides a new insight into the prevention and treatment of genetic diseases.

An Ultrasensitive and Universal Surface Plasmonic Biosensor for Detection of Micropollutants in Aquatic Environments.

Simple yet ultrasensitive and accurate quantification of a variety of analytical targets by virtue of a universal sensing device holds promise to revolutionize environmental monitoring, medical diagnostics, and food safety. Here, we propose a novel optical surface plasmon resonance (SPR) system in which the frequency-shifted light of different polarizations returned the laser cavity to stimulate laser heterodyne feedback interferometry (LHFI), hence amplifying the reflectivity change caused by the refractive index (RI) variations on the gold-coated SPR chip surface. In addition, the s-polarized light was further used as the reference to compensate the noise of the LHFI-amplified SPR system, resulting in nearly 3 orders of magnitude enhancement of RI resolution (5.9 × 10-8 RIU) over the original SPR system (2.0 × 10-5 RIU). By exploiting nucleic acids, antibodies, and receptors as recognition materials, a variety of micropollutants were detected with ultralow detection limits, ranging from a toxic metal ion (Hg2+, 70 ng/L) to a group of commonly occurring biotoxin (microcystins, 3.9 ng microcystin-LR/L) and a class of environmental endocrine disruptors (estrogens, 0.7 ng 17ß-estradiol/L). This sensing platform exhibits several distinct characteristics, including dual improvement of sensitivity and stability and common-path optical construction without needing optical alignment, demonstrating a promising avenue toward environmental monitoring.

A 3,5-dinitropyridin-2yl Substituted Flavonol-based Fluorescent Probe for Rapid Detection of H2S in Water, Foodstuff Samples and Living Cells.

A novel flavonol-based fluorescent probe, Fla-DNT, has been synthesized for the rapid and specific detection of H2S. Fla-DNT exhibits excellent selectivity and anti-interference properties, a short response time (4 min), large Stokes shift (138 nm), and low detection limit (1.357 µM). Upon exposure to H2S, Fla-DNT displays a remarkable increase in fluorescence intensity at 542 nm. Meanwhile, the recognizing site of H2S was predicted through Electrostatic potential and ADCH charges calculations, while the sensing mechanism of H2S was determined via HRMS analysis and DFT calculation. More importantly, the probe owes multiple applications, such as a recovery rate ranging from 92.00 to 102.10% for detecting H2S in water samples, and it can be fabricated into fluorescent strips to track H2S production during food spoilage by tracking color changes, thereby enabling real-time monitoring of food freshness. The bioimaging experiments demonstrate the capability of Fla-DNT to detect both endogenous and exogenous H2S in living cells. These results provide a reliable method and idea for H2S detection in complex environments.

Evaluation of thallium ion as an effective ion in human health using an electrochemical sensor.

Exposure to thallium (Tl), a noxious heavy metal, poses significant health risks to both humans and animals upon ingestion. Therefore, monitoring Tl levels in the environment is crucial to prevent human exposure and reduce the risk of developing severe health problems. This paper presents the development of a highly sensitive Tl ions sensor through surface modification of a glassy carbon electrode with a nanocomposite comprising MnO2 magnetic sepiolite and multi-walled carbon nanotubes (MnO2@Fe3O4/Sep/MWCNT/GCE). Multiple methodologies were employed to assess the performance of the newly developed sensor. By employing square wave anodic stripping voltammetry (SWASV) to optimize the measurement conditions, notable enhancements were observed in the stripping peak currents of Tl (I) on the MnO2@Fe3O4/Sep/MWCNT/GCE surface. The effectiveness of the nanocomposite in facilitating electron transfer between the Tl (I) ions (guest) and the electrode (host) was demonstrated from the enhanced signals observed at the different modified electrode surfaces under optimal conditions. The developed sensor displayed a wide linear range of 0.1-1500 ppb for Tl (I) and a low detection limit of 0.03 ppb for Tl (I). It was found to be selective for Tl (I) ions while remaining unaffected by interfering non-target ions in the presence of the target ions. Despite its simple preparation procedure, the modified electrode exhibited high stability and excellent reproducibility for measuring Tl (I). The outstanding electroanalytical performances of the MnO2@Fe3O4/Sep/MWCNT/GCE electrode enabled its successful use as an ultrasensitive sensor for determining trace amounts of Tl in environmental samples.