Protein Sequencing with Artificial Intelligence: Machine Learning Integrated Phosphorene Nanoslit.

Achieving high throughput protein sequencing at single molecule resolution remains a daunting challenge. Herein, relying on a solid-state 2D phosphorene nanoslit device, an extraordinary biosensor to rapidly identify the key signatures of all twenty amino acids using an interpretable machine learning (ML) model is reported. The XGBoost regression algorithm allows the determination of the transmission function of all twenty amino acids with high accuracy. The resultant ML and DFT studies reveal that it is possible to identify individual amino acids through transmission and current signals readouts with high sensitivity and selectivity. Moreover, we thoroughly compared our results to those from graphene nanoslit and found that the phosphorene nanoslit device can be an ideal candidate for protein sequencing up to a 20-fold increase in transmission sensitivity. The present study facilitates high throughput screening of all twenty amino acids and can be further extended to other biomolecules for disease diagnosis and therapeutic decision making.

Phase behaviors of deeply supercooled bilayer water unseen in bulk water.

Akin to bulk water, water confined to an isolated nanoslit can show a wealth of new 2D phases of ice and amorphous ice, as well as unusual phase behavior. Indeed, 2D water phases, such as bilayer hexagonal ice and monolayer square ice, have been detected in the laboratory, confirming earlier computational predictions. Herein, we report theoretical evidence of a hitherto unreported state, namely, bilayer very low density amorphous ice (BL-VLDA), as well as evidence of a strong first-order transition between BL-VLDA and the BL amorphous ice (BL-A), and a weak first-order transition between BL-VLDA and the BL very low density liquid (BL-VLDL) water. The diffusivity of BL-VLDA is typically in the range of 10-9 cm2/s to 10-10 cm2/s. Similar to bulk (3D) water, 2D water can exhibit two forms of liquid in the deeply supercooled state. However, unlike supercooled bulk water, for which the two forms of liquid can coexist and merge into one at a critical point, the 2D BL-VLDL and BL high-density liquid (BL-HDL) phases are separated by the highly stable solid phase of BL-A whose melting line exhibits the isochore end point (IEP) near 220 K in the temperature-pressure diagram. Above the IEP temperature, BL-VLDL and BL-HDL are indistinguishable. At negative pressures, the metastable BL-VLDL exhibits a spatially and temporally heterogeneous structure induced by dynamic changes in the nanodomains, a feature much less pronounced in the BL-HDL.

Investigation of DNA Hybridization on Nano-Structured Plasmonic Surfaces for Identifying Nasopharyngeal Viruses.

Recently, studies have revealed that human herpesvirus 4 (HHV-4), also known as the Epstein-Barr virus, might be associated with the severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Compared to SARS-CoV-2 infection alone, patients coinfected with SARS-CoV-2 and HHV-4 had higher risks of fever, inflammation, and even death, thus, confirming that HHV-4/SARS-CoV-2 coinfection in patients could benefit from clinical investigation. Although several intelligent devices can simultaneously discern multiple genes related to SARS-CoV-2, most operate via label-based detection, which restricts them from directly measuring the product. In this study, we developed a device that can replicate and detect SARS-CoV-2 and HHV-4 DNA. This device can conduct a duplex polymerase chain reaction (PCR) in a microfluidic channel and detect replicates in a non-labeled manner through a plasmonic-based sensor. Compared to traditional instruments, this device can reduce the required PCR time by 55% while yielding a similar amount of amplicon. Moreover, our device's limit of detection (LOD) reached 100 fg/mL, while prior non-labeled sensors for SARS-CoV-2 detection were in the range of ng/mL to pg/mL. Furthermore, the device can detect desired genes by extracting cells artificially infected with HHV-4/SARS-CoV-2. We expect that this device will be able to help verify HHV-4/SARS-CoV-2 coinfected patients and assist in the evaluation of practical treatment approaches.

Linear-Zero Mode Waveguides for Single-Molecule Fluorescence Observation of Nucleotides in Kinesin-Microtubule Motility Assay.

Single-molecule fluorescence microscopy is a key tool to investigate the chemo-mechanical coupling of microtubule-associated motor proteins, such as kinesin. However, a major limitation of the implementation of single-molecule observation is the concentration of fluorescently labeled molecules. For example, in total internal reflection fluorescence microscopy, the available concentration is of the order of 10 nM. This concentration is much lower than the concentration of adenosine triphosphate (ATP) in vivo, hindering the single-molecule observation of fluorescently labeled ATP hydrolyzed by motor proteins under the physiologically relevant conditions. Here, we provide a method for the use of single-molecule fluorescence microscopy in the presence of ~500 nM of fluorescently labeled ATP. To achieve this, a device equipped with nano-slits is used to confine excitation light into its slits as an expansion of zero-mode waveguides (ZMWs). Conventional ZMWs equip apertures with a diameter smaller than the wavelength of light to suppress background noise from the labeled molecules diffusing outside of the apertures. While they are not compatible with filamentous objects, our linear-ZMWs enable the usage of filamentous objects, such as microtubules. An experiment using linear-ZMWs demonstrated the successful exploration of the interaction between kinesin and ATP using single-molecule fluorescence microscopy.

Enhancing and Broadening the Photoresponse of Monolayer MoS2 Based on Au Nanoslit Array.

Two-dimensional molybdenum disulfide (MoS2), featuring unique optoelectronic properties, has attracted tremendous interest in developing novel photodetection devices. However, the limited light absorption and small carrier transport rate of the monolayer MoS2 result in low photoresponse, and the large band gap limits its detection range in the visible region. In this study, we propose a nanoslit array-MoS2 hybrid device architecture with enhanced and broadened photoresponse. The nanoslit array can localize free-space light to achieve strong interactions with MoS2, and acts as the channel to improve charge transport. As a result, the Au nanoslit array-MoS2 hybrid detector exhibits a nearly 100-fold increase in photocurrent compared to the pure MoS2 device. More importantly, the hybrid device can broaden the photoresponse to the optical communication band of 1550 nm which is lower than the band gap of MoS2, by efficiently utilizing the hot carriers generated by the Au nanoslits. The experimental results are supported by both theoretical analysis and numerical simulation. Since our demonstration leverages the engineering of the hybrid photodetectors with metal nanostructures rather than semiconductor materials, it should be universal and applicable to other devices for broadband, high-efficiency photoelectric conversion.

Continuous polymerase chain reaction microfluidics integrated with a gold-capped nanoslit sensing chip for Epstein-Barr virus detection.

We present the first combination of a microfluidic polymerase chain reaction (PCR) with a gold nanoslit-based surface plasmon resonance (SPR) sensor for detecting the DNA sequence of latent membrane protein 1 (LMP1). The PCR microchannel was produced through a laser scribing technique, and the SPR nanoslit chip was manufactured via hot-embossing nanoimprinting lithography. Afterward, the LMP1 DNA probe was adsorbed onto the SPR chip of the integrated device through electrostatic interactions for further detection. The device can complete the analytical procedure in around 36 min, while the traditional machine requires 105 min to achieve similar signals under the same PCR thermal cycles. The calibration curve with serially diluted LMP1 DNA exhibited the accuracy (R2 > 0.99) and sensitivity (limit of detection: ∼10-11 g/mL) of the device. Moreover, extracted DNA from Epstein-Barr virus (EBV)-positive cells were directly detected through the integrated chip. In brief, this all-in-one chip can amplify gene fragments at the front-end and detect them at the back-end, decreasing the time required for the analysis without compromising accuracy or sensitivity. We believe this label-free, real-time, low-cost device has enormous potential for rapid detection of various viruses, such as EBV and COVID-19.

Numerical Study on Enhanced Line Focusing via Buried Metallic Nanowire Assisted Binary Plate.

Line focusing, which collects light into a line rather than a single point, has an advantage on variable fields such as machining and imaging. The 1-dimensional metallic zone plate is one of the candidates for line focusing, which is ultra-thin and simple to fabricate. Metallic nano-slits can replace the metal blocked region to increase the efficiency, however, the efficiency and stability are still low. Therefore, this paper proposes a structure with an additional dielectric layer to protect the metallic nano-slit layer-a buried metallic wire structure-and verify the idea based on numerical simulations. Two structures are proposed. In terms of stability, a flat surface structure is proposed and a corrugated surface structure with a consistent thickness with the nano-slit is proposed which has low fabrication difficulty. The optimization of the buried wire structure and performance after applying the buried wire structure to the dual-line focusing plate is calculated by numerical simulation. Finally, it was shown that the electric field intensity was 2.13 times greater.

Multichannel nanoplasmonic platform for imidacloprid and fipronil residues rapid screen detection.

In recent years, imidacloprid and fipronil have been reported to harm beneficial insects, such as honey bees, and to potentially pose risks to mammals, including humans. Considering their widespread use and potential minimum toxic range from 10 ppb to 1 ppm (species dependent), a simple, rapid, sensitive, and reliable method for screening and detection is urgently needed. Here, we present a surface plasmon resonance (SPR)-based nanoplasmonic chip integrated with a multichannel spectral imaging system to detect ecosystem-harming pesticides. The pre-modification of the designed mercapto-haptens reduced detection time to 2.5 h. Moreover, owing to the multichannel configuration, it was possible to introduce an internal standard analytical method to effectively reduce matrix interference in real samples; thus, the concentration of the target pesticide could be determined more precisely. The strong linearity of the spiked sample test results indicated high accuracy in quantifying target pesticides. Considering the limit of detection (~10 ppb), the cutoffs for detection and quantification were set at 15 and 45 ppb, respectively, and were used as the detection criteria. The detection results of the blind tests of real samples were also compared with those of liquid chromatography electrospray ionization tandem mass spectrometry (standard method) and were highly consistent. The custom-made integrated SPR system allows much simpler, label-free, high-throughput, and reliable on-site identification and quantification of imidacloprid and fipronil. All test results validated the platform's capability in the on-site rapid screening and detection of pesticide residues at the parts per billion and parts per million levels.

Plasmonic Nanoslit Arrays Fabricated by Serial Bideposition: Optical and Surface-Enhanced Raman Scattering Study.

Recently, studies have been carried out to combine surface-enhanced Raman spectroscopy substrates that are based on either localized surface plasmon or surface plasmon polariton structures. By combining these two systems, the individual drawbacks of each can be overcome. However, the manufacturing methods involved so far are sophisticated, labor-intensive, expensive, and technically demanding. We propose a facile method for the fabrication of a flexible plasmonic nanoslit surface-enhanced Raman scattering (SERS) sensor. We utilized the pattern on periodic optical disks as an inexpensive substitute for printing the periodic pattern on polydimethylsiloxane with soft imprint lithography. The Ag nanoslits were fabricated by serial bideposition using the dynamic oblique angle deposition technique. The nanoslit structures were physically and optically characterized, and the experimental results were compared to the results of the numerical simulation: Monte Carlo and finite-difference time-domain simulation. The AgNS samples showed excellent SERS performance with an enhancement factor of ∼105 and a limit of detection of 5 × 10-7 g/mL for a Rhodamine 6G solution. Their biosensing capability was demonstrated by the sensing of bilirubin.

Investigation on Super-Resolution Focusing Performance of a TE-Polarized Nanoslit-Based Two-Dimensional Lens.

Conventional optics suffer from the diffraction limit. Our recent work has predicted a nanoslit-based two-dimensional (2D) lens with transverse-electric (TE) polarized design that is capable of realizing the super-resolution focusing of light beyond the diffraction limit in the quasi-far field. Furthermore, the super-resolution capability can be kept in a high-refractive-index dielectric over a wide wavelength range from ultraviolet to visible light. Here, we systematically investigate the influence of various factors on the super-resolution focusing performance of the lens. Factors such as lens aperture, focal length and nanoslit length are considered. In particular, the influence of nanoslit length on lens focusing was ignored in the previous reports about nanoslit-based 2D lenses, since nanoslit length was assumed to be infinite. The numerical results using the finite-difference time-domain (FDTD) method demonstrate that the super-resolution focusing capability of a nanoslit-based 2D lens increases with the lens aperture and reduces with the increase of the lens focal length. On the other hand, it is notable that the length of the lens focus is not equal to but smaller than that of the nanoslits. Therefore, in order to achieve a desired focus length, a lens should be designed with longer nanoslits.

Simultaneous Observation of Kinesin-Driven Microtubule Motility and Binding of Adenosine Triphosphate Using Linear Zero-Mode Waveguides.

Single-molecule fluorescence observation of adenosine triphosphate (ATP) is a powerful tool to elucidate the chemomechanical coupling of ATP with a motor protein. However, in total internal reflection fluorescence microscopy (TIRFM), available ATP concentration is much lower than that in the in vivo environment. To achieve single-molecule observation with a high signal-to-noise ratio, zero-mode waveguides (ZMWs) are utilized even at high fluorescent molecule concentrations in the micromolar range. Despite the advantages of ZMWs, the use of cytoskeletal filaments for single-molecule observation has not been reported because of difficulties in immobilization of cytoskeletal filaments in the cylindrical aperture of ZMWs. Here, we propose linear ZMWs (LZMWs) to visualize enzymatic reactions on cytoskeletal filaments, specifically kinesin-driven microtubule motility accompanied by ATP binding/unbinding. Finite element method simulation revealed excitation light confinement in a 100 nm wide slit of LZMWs. Single-molecule observation was then demonstrated with up to 1 µM labeled ATP, which was 10-fold higher than that available in TIRFM. Direct observation of binding/unbinding of ATP to kinesins that propel microtubules enabled us to find that a significant fraction of ATP molecules bound to kinesins were dissociated without hydrolysis. This highlights the advantages of LZMWs for single-molecule observation of proteins that interact with cytoskeletal filaments such as microtubules, actin filaments, or intermediate filaments.

Transmission surface plasmon resonance techniques and their potential biosensor applications.

Transmission surface plasmon resonance (TSPR) is an unusual extraordinary optical transmission that is more transparent at certain wavelengths than expected by classical theory. The three main plasmonic structures that providing this phenomenon are nanohole arrays, diffraction gratings, and nanoslit arrays. This extraordinary optical transmission phenomenon is produced as a result of surface plasmon excitations. The shifting in TSPR responses upon changing of dielectric environment at the surface of a metallic film was observed. After TSPR was discovered from metallic nanohole arrays in 1998, the number of papers about this topic rapidly increased. In the 20 years since, TSPR has been utilized to improve the detection limits, sensitivity, selectivity, and dynamic range of biosensing devices, resulting in them having greater potential for commercialization. This review gives a broad overview of the TSPR phenomenon, the development of this technique, and the typical experimental setups used to acquire TSPR signals; it also describes how they are applied in the field of research into biosensors.

A slanted-nanoaperture metal lens: subdiffraction-limited focusing of light in the intermediate field region.

Diffraction of light limits the resolution of beam focusing with conventional lenses, as dictated by the Abbe limit, that is, approximately half the wavelength. Numerous techniques have been explored to overcome this limit. One of the most intensively explored approaches is to design a lens that operates in the near-field region, that is, with a focal length on the order of 10 nm, where evanescent fields can carry and project large in-plane wave-vectors (greater than free-space wave-vectors) to a focal plane. From a practical perspective, however, the requirement of such an ultra-short focal length puts too much constraint, since much longer focal length is commonly desired for intermediate or far-field operation. Here we report a method to beat the Abbe limit while operating with focal length greater than wavelength λ. Our approach is to tailor the radiation patterns of nanoaperture transmission by tilting aperture axes away from the surface of a metal film such that each slanted aperture transmits a highly directed, tilt-oriented beam onto a common focal point carrying maximal in-plane wave-vector components. The proposed nanoaperture array lens was fabricated by forming tilted nanoslits in a Ag, Al, or Cr film. We demonstrate minimal spot size of λ/3 (210-nm or 110-nm full-width half-maximum at λ = 633 nm or 325 nm, respectively) with 1-4λ focal length in air, beating the Abbe limit.

Electroosmotic Flow of Viscoelastic Fluid in a Nanoslit.

The electroosmotic flow (EOF) of viscoelastic fluid in a long nanoslit is numerically studied to investigate the rheological property effect of Linear Phan-Thien-Tanner (LPTT) fluid on the fully developed EOF. The non-linear Poisson-Nernst-Planck equations governing the electric potential and the ionic concentration distribution within the channel are adopted to take into account the effect of the electrical double layer (EDL), including the EDL overlap. When the EDL is not overlapped, the velocity profiles for both Newtonian and viscoelastic fluids are plug-like and increase sharply near the charged wall. The velocity profile resembles that of pressure-driven flow when the EDL is overlapped. Regardless of the EDL thickness, apparent increase of velocity is obtained for viscoelastic fluid of larger Weissenberg number compared to the Newtonian fluid, indicating the shear thinning behavior of the LPTT fluid. The effect of the Weissenberg number on the velocity distribution is less significant as the degree of EDL overlapping increases, due to the overall decrease of the shear rate. The increase (decrease) of polymer extensibility (viscosity ratio) also enhances the EOF of viscoelastic fluid.

Aptamer-functionalized nanoparticles for surface immobilization-free electrochemical detection of cortisol in a microfluidic device.

Monitoring the periodic diurnal variations in cortisol from small volume samples of serum or saliva is of great interest, due to the regulatory role of cortisol within various physiological functions and stress symptoms. Current detection assays are immunologically based and require cumbersome antibody immobilization chemistries, thereby limiting the assay versatility, kinetics, and reproducibility. We present a quantitative aptamer-based detection methodology for cortisol that does not require target labeling, capture probe immobilization on the detection surface or wash steps prior to readout. Using a recognition system of aptamer functionalized gold nanoparticles pre-bound with electro-active triamcinolone, the cortisol level is detected based on its competitive binding to the aptamer by following signal from the displaced triamcinolone using square wave voltammetry at patterned graphene-modified electrodes in a microfluidic or nanoslit device. Due to the 3D analyte diffusion profile at the aptamer interface and the ability to enhance the surface area for cortisol capture, this assay shows signal linearity over a five-log analyte concentration range (10 µg/mL to 30 pg/mL) and exhibits rapid binding kinetics with cortisol versus other glucocorticoids, as apparent from the absence of interferences from estradiol, testosterone and progesterone. The assay is carried out within the biologically relevant range for glucocorticoids in serum and saliva matrices, and benchmarked versus ELISA and radioimmunoassays. Based on absence of cumbersome surface immobilization and wash steps for carrying out this assay, its quantitative signal characteristics and its ability to resist interferences from other glucocorticoids, we envision its application towards routine monitoring of cortisol within bio-fluids.