Angular-Inertia Regulated Stable and Nanoscale Sensing of Single Molecules Using Nanopore-In-A-Tube.

Nanopore is commonly used for high-resolution, label-free sensing, and analysis of single molecules. However, controlling the speed and trajectory of molecular translocation in nanopores remains challenging, hampering sensing accuracy. Here, the study proposes a nanopore-in-a-tube (NIAT) device that enables decoupling of the current signal detection from molecular translocation and provides precise angular inertia-kinetic translocation of single molecules through a nanopore, thus ensuring stable signal readout with high signal-to-noise ratio (SNR). Specifically, the funnel-shaped silicon nanopore, fabricated at a 10-nm resolution, is placed into a centrifugal tube. A light-induced photovoltaic effect is utilized to achieve a counter-balanced state of electrokinetic effects in the nanopore. By controlling the inertial angle and centrifugation speed, the angular inertial force is harnessed effectively for regulating the translocation process with high precision. Consequently, the speed and trajectory of the molecules are able to be adjusted in and around the nanopore, enabling controllable and high SNR current signals. Numerical simulation reveals the decisive role of inertial angle in achieving uniform translocation trajectories and enhancing analyte-nanopore interactions. The performance of the device is validated by discriminating rigid Au nanoparticles with a 1.6-nm size difference and differentiating a 1.3-nm size difference and subtle stiffness variations in flexible polyethylene glycol molecules.

Automated Uniform Spheroid Generation Platform for High Throughput Drug Screening Process.

Three-dimensional (3D) spheroid models are crucial for cancer research, offering more accurate insights into tumour biology and drug responses than traditional 2D cell cultures. However, inconsistent and low-throughput spheroid production has hindered their application in drug screening. Here, we present an automated high-throughput platform for a spheroid selection, fabrication, and sorting system (SFSS) to produce uniform gelatine-encapsulated spheroids (GESs) with high efficiency. SFSS integrates advanced imaging, analysis, photo-triggered fabrication, and microfluidic sorting to precisely control spheroid size, shape, and viability. Our data demonstrate that our SFSS can produce over 50 GESs with consistent size and circularity in 30 min with over 97% sorting accuracy while maintaining cell viability and structural integrity. We demonstrated that the GESs can be used for drug screening and potentially for various assays. Thus, the SFSS could significantly enhance the efficiency of generating uniform spheroids, facilitating their application in drug development to investigate complex biological systems and drug responses in a more physiologically relevant context.

Coupled photothermal vortices for capture, sorting, and transportation of particles.

Optofluidic techniques have evolved as a prospering strategy for microparticle manipulation via fluid. Unfortunately, there is still a lack of manipulation with simple preparation, easy operation, and multifunctional integration. In this Letter, we present an optofluidic device based on a graphite oxide (GO)-coated dual-fiber structure for multifunctional particle manipulation. By changing the optical power and the relative distance of the fibers, the system can excite thermal fluidic vortices with three inter-coupled states, namely uncoupled, partially coupled and completely coupled states, and therefore can realize capture, sorting, and transportation of the target particles. We conduct a numerical analysis of the whole system, and the results are consistent with the experimental phenomena. This versatile device can be utilized to manipulate target particles in complex microscopic material populations with the advantages of flexible operation, user-friendly control, and low cost.

In-tube micro-pyramidal silicon nanopore for inertial-kinetic sensing of single molecules.

Electrokinetic force has been the major choice for driving the translocation of molecules through a nanopore. However, the use of this approach is limited by an uncontrollable translocation speed, resulting in non-uniform conductance signals with low conformational sensitivity, which hinders the accurate discrimination of the molecules. Here, we show the use of inertial-kinetic translocation induced by spinning an in-tube micro-pyramidal silicon nanopore fabricated using photovoltaic electrochemical etch-stop technique for biomolecular sensing. By adjusting the kinetic properties of a funnel-shaped centrifugal force field while maintaining a counter-balanced state of electrophoretic and electroosmotic effect in the nanopore, we achieved regulated translocation of proteins and obtained stable signals of long and adjustable dwell times and high conformational sensitivity. Moreover, we demonstrated instantaneous sensing and discrimination of molecular conformations and longitudinal monitoring of molecular reactions and conformation changes by wirelessly measuring characteristic features in current blockade readouts using the in-tube nanopore device.

Advancing MicroRNA Detection: Enhanced Biotin-Streptavidin Dual-Mode Phase Imaging Surface Plasmon Resonance Aptasensor.

MicroRNAs (miRNAs) are novel tumor biomarkers owing to their important physiological functions in cell communication and the progression of multiple diseases. Due to the small molecular weight, short sequence length, and low concentration levels of miRNA, miRNA detection presents substantial challenges, requiring the advancement of more refined and sensitive techniques. There is an urgent demand for the development of a rapid, user-friendly, and sensitive miRNA analysis method. Here, we developed an enhanced biotin-streptavidin dual-mode phase imaging surface plasmon resonance (PI-SPR) aptasensor for sensitive and rapid detection of miRNA. Initially, we evaluated the linear sensing range for miRNA detection across two distinct sensing modalities and investigated the physical factors that influence the sensing signal in the aptamer-miRNA interaction within the PI-SPR aptasensor. Then, an enhanced biotin-streptavidin amplification strategy was introduced in the PI-SPR aptasensor, which effectively reduced the nonspecific adsorption by 20% and improved the limit of detection by 548 times. Furthermore, we have produced three types of tumor marker chips, which utilize the rapid sensing mode (less than 2 min) of PI-SPR aptasensor to achieve simultaneous detection of multiple miRNA markers in the serum from clinical cancer patients. This work not only developed a new approach to detect miRNA in different application scenarios but also provided a new reference for the application of the biotin-streptavidin amplification system in the detection of other small biomolecules.

RSPSSL: A novel high-fidelity Raman spectral preprocessing scheme to enhance biomedical applications and chemical resolution visualization.

Raman spectroscopy has tremendous potential for material analysis with its molecular fingerprinting capability in many branches of science and technology. It is also an emerging omics technique for metabolic profiling to shape precision medicine. However, precisely attributing vibration peaks coupled with specific environmental, instrumental, and specimen noise is problematic. Intelligent Raman spectral preprocessing to remove statistical bias noise and sample-related errors should provide a powerful tool for valuable information extraction. Here, we propose a novel Raman spectral preprocessing scheme based on self-supervised learning (RSPSSL) with high capacity and spectral fidelity. It can preprocess arbitrary Raman spectra without further training at a speed of ~1 900 spectra per second without human interference. The experimental data preprocessing trial demonstrated its excellent capacity and signal fidelity with an 88% reduction in root mean square error and a 60% reduction in infinite norm ([Formula: see text]) compared to established techniques. With this advantage, it remarkably enhanced various biomedical applications with a 400% accuracy elevation (ΔAUC) in cancer diagnosis, an average 38% (few-shot) and 242% accuracy improvement in paraquat concentration prediction, and unsealed the chemical resolution of biomedical hyperspectral images, especially in the spectral fingerprint region. It precisely preprocessed various Raman spectra from different spectroscopy devices, laboratories, and diverse applications. This scheme will enable biomedical mechanism screening with the label-free volumetric molecular imaging tool on organism and disease metabolomics profiling with a scenario of high throughput, cross-device, various analyte complexity, and diverse applications.

Highly-Adaptable Optothermal Nanotweezers for Trapping, Sorting, and Assembling across Diverse Nanoparticles.

Optical manipulation of various kinds of nanoparticles is vital in biomedical engineering. However, classical optical approaches demand higher laser power and are constrained by diffraction limits, necessitating tailored trapping schemes for specific nanoparticles. They lack a universal and biocompatible tool to manipulate nanoparticles of diverse sizes, charges, and materials. Through precise modulation of diffusiophoresis and thermo-osmotic flows in the boundary layer of an optothermal-responsive gold film, highly adaptable optothermal nanotweezers (HAONTs) capable of manipulating a single nanoparticle as small as sub-10 nm are designed. Additionally, a novel optothermal doughnut-shaped vortex (DSV) trapping strategy is introduced, enabling a new mode of physical interaction between cells and nanoparticles. Furthermore, this versatile approach allows for the manipulation of nanoparticles in organic, inorganic, and biological forms. It also offers versatile function modes such as trapping, sorting, and assembling of nanoparticles. It is believed that this approach holds the potential to be a valuable tool in fields such as synthetic biology, optofluidics, nanophotonics, and colloidal science.

Deep-qGFP: A Generalist Deep Learning Assisted Pipeline for Accurate Quantification of Green Fluorescent Protein Labeled Biological Samples in Microreactors.

Absolute quantification of biological samples provides precise numerical expression levels, enhancing accuracy, and performance for rare templates. Current methodologies, however, face challenges-flow cytometers are costly and complex, whereas fluorescence imaging, relying on software or manual counting, is time-consuming and error-prone. It is presented that Deep-qGFP, a deep learning-aided pipeline for the automated detection and classification of green fluorescent protein (GFP) labeled microreactors, enables real-time absolute quantification. This approach achieves an accuracy of 96.23% and accurately measures the sizes and occupancy status of microreactors using standard laboratory fluorescence microscopes, providing precise template concentrations. Deep-qGFP demonstrates remarkable speed, quantifying over 2000 microreactors across ten images in just 2.5 seconds, with a dynamic range of 56.52-1569.43 copies µL-1 . The method demonstrates impressive generalization capabilities, successfully applied to various GFP-labeling scenarios, including droplet-based, microwell-based, and agarose-based applications. Notably, Deep-qGFP is the first all-in-one image analysis algorithm successfully implemented in droplet digital polymerase chain reaction (PCR), microwell digital PCR, droplet single-cell sequencing, agarose digital PCR, and bacterial quantification, without requiring transfer learning, modifications, or retraining. This makes Deep-qGFP readily applicable in biomedical laboratories and holds potential for broader clinical applications.

Label-free biosensing with singular-phase-enhanced lateral position shift based on atomically thin plasmonic nanomaterials.

Rapid plasmonic biosensing has attracted wide attention in early disease diagnosis and molecular biology research. However, it was still challenging for conventional angle-interrogating plasmonic sensors to obtain higher sensitivity without secondary amplifying labels such as plasmonic nanoparticles. To address this issue, we developed a plasmonic biosensor based on the enhanced lateral position shift by phase singularity. Such singularity presents as a sudden phase retardation at the dark point of reflection from resonating plasmonic substrate, leading to a giant position shift on reflected beam. Herein, for the first time, the atomically thin layer of Ge2Sb2Te5 (GST) on silver nanofilm was demonstrated as a novel phase-response-enhancing plasmonic material. The GST layer was not only precisely engineered to singularize phase change but also served as a protective layer for active silver nanofilm. This new configuration has achieved a record-breaking largest position shift of 439.3 µm measured in calibration experiments with an ultra-high sensitivity of 1.72 × 108 nm RIU-1 (refractive index unit). The detection limit was determined to be 6.97 × 10-7 RIU with a 0.12 µm position resolution. Besides, a large figure of merit (FOM) of 4.54 × 1011 µm (RIU∙°)-1 was evaluated for such position shift interrogation, enabling the labelfree detection of trace amounts of biomolecules. In targeted biosensing experiments, the optimized sensor has successfully detected small cytokine biomarkers (TNF-α and IL-6) with the lowest concentration of 1 × 10-16 M. These two molecules are the key proinflammatory cancer markers in clinical diagnosis, which cannot be directly screened by current clinical techniques. To further validate the selectivity of our sensing systems, we also measured the affinity of integrin binding to arginylglycylaspartic acid (RGD) peptide (a key protein interaction in cell adhesion) with different Mn2+ ion concentrations, ranging from 1 nM to 1 mM.

CRISPR-powered optothermal nanotweezers: Diverse bio-nanoparticle manipulation and single nucleotide identification.

Optothermal nanotweezers have emerged as an innovative optical manipulation technique in the past decade, which revolutionized classical optical manipulation by efficiently capturing a broader range of nanoparticles. However, the optothermal temperature field was merely employed for in-situ manipulation of nanoparticles, its potential for identifying bio-nanoparticles remains largely untapped. Hence, based on the synergistic effect of optothermal manipulation and CRIPSR-based bio-detection, we developed CRISPR-powered optothermal nanotweezers (CRONT). Specifically, by harnessing diffusiophoresis and thermo-osmotic flows near the substrate upon optothermal excitation, we successfully trapped and enriched DNA functionalized gold nanoparticles, CRISPR-associated proteins, as well as DNA strands. Remarkably, we built an optothermal scheme for enhancing CRISPR-based single-nucleotide polymorphism (SNP) detection at single molecule level, while also introducing a novel CRISPR methodology for observing nucleotide cleavage. Therefore, this innovative approach has endowed optical tweezers with DNA identification ability in aqueous solution which was unattainable before. With its high specificity and feasibility for in-situ bio-nanoparticle manipulation and identification, CRONT will become a universal tool in point-of-care diagnosis, biophotonics, and bio-nanotechnology.

Quasi-phase extraction-based surface plasmon resonance imaging method for coffee ring effect monitoring and biosensing.

Wavelength interrogation surface plasmon resonance imaging (WSPRi) sensing has unique advantages in high-throughput imaging detection. The refractive index resolution (RIR) of WSPRi is limited to the order of 10-6 RIU. This paper demonstrates a novel WSPRi sensing system with a wavelength scanning device of an acousto-optic tunable filter (AOTF) and a low-cost speckle-free SPR excitation source of a halogen lamp. We developed a sensitive quasi-phase extraction method for data processing. The new technique achieved an RIR of 8.84×10-7 RIU, which is the first WSPRi system that has an RIR in the order of 10-7 RIU. Moreover, we performed a real-time recording of the formation of the coffee ring effect during brine evaporation and enhanced the biosensor performance of SPR for the first time. We believe the higher RIR and accuracy of the system will benefit more potential applications toward exploring the biomolecules' behaviors in biological and biochemistry studies.

Unraveling the complexity of Optical Coherence Tomography image segmentation using machine and deep learning techniques: A review.

Optical Coherence Tomography (OCT) is an emerging technology that provides three-dimensional images of the microanatomy of biological tissue in-vivo and at micrometer-scale resolution. OCT imaging has been widely used to diagnose and manage various medical diseases, such as macular degeneration, glaucoma, and coronary artery disease. Despite its wide range of applications, the segmentation of OCT images remains difficult due to the complexity of tissue structures and the presence of artifacts. In recent years, different approaches have been used for OCT image segmentation, such as intensity-based, region-based, and deep learning-based methods. This paper reviews the major advances in state-of-the-art OCT image segmentation techniques. It provides an overview of the advantages and limitations of each method and presents the most relevant research works related to OCT image segmentation. It also provides an overview of existing datasets and discusses potential clinical applications. Additionally, this review gives an in-depth analysis of machine learning and deep learning approaches for OCT image segmentation. It outlines challenges and opportunities for further research in this field.

Rapid Prototyping of Multi-Functional and Biocompatible Parafilm®-Based Microfluidic Devices by Laser Ablation and Thermal Bonding.

In this paper, we report a simple, rapid, low-cost, biocompatible, and detachable microfluidic chip fabrication method for customized designs based on Parafilm®. Here, Parafilm® works as both a bonding agent and a functional membrane. Its high ultimate tensile stress (3.94 MPa) allows the demonstration of high-performance actuators such as microvalves and micropumps. By laser ablation and the one-step bonding of multiple layers, 3D structured microfluidic chips were successfully fabricated within 2 h. The consumption time of this method (~2 h) was 12 times less than conventional photolithography (~24 h). Moreover, the shear stress of the PMMA-Parafilm®-PMMA specimens (0.24 MPa) was 2.13 times higher than that of the PDMS-PDMS specimens (0.08 MPa), and 0.56 times higher than that of the PDMS-Glass specimens (0.16 MPa), showing better stability and reliability. In this method, multiple easily accessible materials such as polymethylmethacrylate (PMMA), PVC, and glass slides were demonstrated and well-incorporated as our substrates. Practical actuation devices that required high bonding strength including microvalves and micropumps were fabricated by this method with high performance. Moreover, the biocompatibility of the Parafilm®-based microfluidic devices was validated through a seven-day E. coli cultivation. This reported fabrication scheme will provide a versatile platform for biochemical applications and point-of-care diagnostics.

Wavelength sequential selection technique for high-throughput multi-channel phase interrogation surface plasmon resonance imaging sensing.

Phase interrogation surface plasmon resonance (P-SPR) biosensors have the highest sensitivity among different types of surface plasmon resonance (SPR) biosensors. However, P-SPR sensors have small dynamic detection range and complex device configuration. To solve these two problems, we designed a multi-channel P-SPR imaging (mcP-SPRi) sensing platform based on a common-path ellipsometry scheme. A wavelength sequential selection (WSS) technique for P-SPRi sensing is developed to select the optimal sensing wavelengths according to different refractive indexes (RIs) of the samples, so the inconsistency of SPR signal response for different biomolecule types caused by the small dynamic detection range is eliminated. And a dynamic detection range of 3.7×10-3 RIU is achieved, which is the largest among the current mcP-SPRi biosensors. Remarkably, the individual SPR phase image acquisition time has been greatly reduced to 1s by using WSS method instead of whole spectrum scanning, which enables the high-throughput mcP-SPRi sensing.

An Integrated ddPCR Lab-on-a-Disc Device for Rapid Screening of Infectious Diseases.

Digital droplet PCR (ddPCR) is a powerful amplification technique for absolute quantification of viral nucleic acids. Although commercial ddPCR devices are effective in the lab bench tests, they cannot meet current urgent requirements for on-site and rapid screening for patients. Here, we have developed a portable and fully integrated lab-on-a-disc (LOAD) device for quantitively screening infectious disease agents. Our designed LOAD device has integrated (i) microfluidics chips, (ii) a transparent circulating oil-based heat exchanger, and (iii) an on-disc transmitted-light fluorescent imaging system into one compact and portable box. Thus, droplet generation, PCR thermocycling, and analysis can be achieved in a single LOAD device. This feature is a significant attribute for the current clinical application of disease screening. For this custom-built ddPCR setup, we have first demonstrated the loading and ddPCR amplification ability by using influenza A virus-specific DNA fragments with different concentrations (diluted from the original concentration to 107 times), followed by analyzing the droplets with an external fluorescence microscope as a standard calibration test. The measured DNA concentration is linearly related to the gradient-dilution factor, which validated the precise quantification for the samples. In addition to the calibration tests using DNA fragments, we also employed this ddPCR-LOAD device for clinical samples with different viruses. Infectious samples containing five different viruses, including influenza A virus (IAV), respiratory syncytial virus (RSV), varicella zoster virus (VZV), Zika virus (ZIKV), and adenovirus (ADV), were injected into the device, followed by analyzing the droplets with an external fluorescence microscope with the lowest detected concentration of 20.24 copies/µL. Finally, we demonstrated the proof-of-concept detection of clinical samples of IAV using the on-disc fluorescence imaging system in our fully integrated device, which proves the capability of this device in clinical sample detection. We anticipate that this integrated ddPCR-LOAD device will become a flexible tool for on-site disease detection.

Real-Time Detection of Circulating Tumor Cells in Bloodstream Using Plasmonic Fiber Sensors.

Circulating tumor cells (CTCs) are single cancer cells or cancer cell clusters that are present in the circulatory system. Assessing CTC levels in patients can aid in the early detection of cancer metastasis and is essential for the purposes of accurate cancer prognosis. However, current in vitro blood tests are limited by the insufficient blood samples and low concentration levels of CTCs, which presents a major challenge for practical biosensing devices. In this work, we propose the first surface plasmon resonance (SPR) fiber probe to work intravenously, which offers a real-time detection of CTCs in bloodstreams. By exposing the protein-functionalized fiber probe to circulating blood, a continuous capture of CTCs ensures a constant increase in enrichment and hence greatly enhances enumeration accuracy. The performance of our plasmonic fiber probe was demonstrated to specifically detect Michigan Cancer Foundation-7 (MCF-7) breast cancer cells in flowing whole mouse blood. Further, a detection limit of ~1.4 cells per microliter was achieved by using an epithelial cell adhesion molecule (EpCAM) antibody-based receptor layer and a 15 minute enrichment period. This pilot study validates real-time CTC detection directly in the bloodstream by using plasmonic fiber probes, which exhibit promising clinical potential for in vivo diagnostic tests involving low concentration biomarkers in circulating blood.

Highly Sensitive Plasmonic Biosensors with Precise Phase Singularity Coupling on the Metastructures.

In this paper, we demonstrated the ability of a plasmonic metasensor to detect ultra-low refractive index changes (in the order of ∆n = 10-10 RIU), using an innovative phase-change material, vanadium dioxide (VO2), as the sensing layer. Different from current cumbersome plasmonic biosensing setups based on optical-phase-singularity measurement, our phase signal detection is based on the direct measurement of the phase-related lateral position shift (Goos-Hänchen) at the sensing interface. The high sensitivity (1.393 × 108 µm/RIU for ∆n = 10-10 RIU), based on the Goos-Hänchen lateral shift of the reflected wave, becomes significant when the sensor is excited at resonance, due to the near-zero reflectivity dip, which corresponds to the absolute dark point (lower than 10-6). GH shifts in the order of 2.997 × 103 µm were obtained using the optimal metasurface configuration. The surface plasmon resonance (SPR) curves (reflectivity, phase, GH) and electromagnetic simulations were derived using the MATLAB programming algorithm (by the transfer matrix method) and Comsol modeling (by finite element analysis), respectively. These results will provide a feasible way for the detection of cancer biomarkers.

A high spatial resolution osteogenic differentiation in human mesenchymal stem cells induced by femtosecond laser.

A variety of physical and chemical methods have been developed in research laboratories for the induction of stem cell differentiation. However, the use of exogenous chemicals and materials may limit their widespread utility in clinics. To develop a clean and precise induction approach with minimal invasion, we reported here that 1-second stimulation by a tightly focused femtosecond laser (fsL) (140 mW/µm2 , 200 fs) can modulate the signaling systems in human mesenchymal cells, such as intracellular calcium and reactive oxygen species. Upon stimulation on an automatic platform, hMSCs were found to express osteoblastic markers and form calcium-rich deposits. Moreover, tissue mineralization was observed when the fsL-illuminated hMSCs were ectopically transplanted into nude mice. Collectively, we described a novel and non-contact optical stimulation method for cell differentiation with high spatiotemporal resolution.

Corrigendum: High-Sensitive Surface Plasmon Resonance Imaging Biosensor Based on Dual-Wavelength Differential Method.

[This corrects the article DOI: 10.3389/fchem.2021.801355.].

Optothermophoretic flipping method for biomolecule interaction enhancement.

The widely used surface-based biomolecule sensing scheme has greatly facilitated the investigation of protein-protein interactions in lab-on-a-chip microfluidic systems. However, in most biosensing schemes, the interactions are driven in a passive way: The overall sensing time and sensitivity are totally dependent on the Brownian diffusion process, which has greatly hindered their efficiency, especially at low concentration levels or single-molecule analysis. To break this limitation, we developed an all-optical active method termed optothermophoretic flipping (OTF). It is the first temporal modulated method that biomolecules were enriched and pushed to their counterparts for effective contact via a flipped thermophoresis. As a proof-of-concept experiment, we tested its performance via antibody-antigen binding on a surface plasmon resonance imaging (SPRi) platform. Compared with the interaction solely based on Brownian diffusion, we achieved a 23.6-fold sensitivity increment in biomolecule interactions sensing. This method has opened new opportunities for various biosensing platforms that require high-sensitivity in colloidal sciences and biochemical studies.