Rapid Detection of Virus Nucleic Acid via Isothermal Amplification on Plasmonic Enhanced Digitizing Biosensor.

Rapid detection for infectious diseases is highly demanded in diagnosis and infection prevention. In this work, we introduced a plasmonic enhanced digitizing biosensor for the rapid detection of nucleic acids. The sensor successfully achieved the detection of loop-mediated isothermal amplification for the hepatitis virus in this work.

Microfluidic compartmentalization to identify gene biomarkers of infection.

Infectious diseases caused by pathogens, such as SARS-COV, H7N9, severe fever with thrombocytopenia syndrome virus, and human immunodeficiency virus, have fatal outcomes with common features of severe fever and subsequent bacterial invasion progressing to multiorgan failure. Gene biomarkers are promising to distinguish specific infections from others with similar presenting symptoms for the prescription of correct therapeutics, preventing pandemics. While routine laboratory methods based on polymerase chain reaction (PCR) to measure gene biomarkers have provided highly sensitive and specific viral detection techniques over the years, they are still hampered by their precision and resource intensity precluding their point-of-care use.

Nanoplasmon-enhanced drop-screen for high throughput single-cell nucleocytoplasmic miRNA profiling.

Cell nucleocytoplasmic profiles of microRNAs (miRNAs) are critical to determining a single cell's essential functionalities, such as cellular transcription, nucleus export and degradation, which gives a comprehensive view of cellular processes. Despite the importance of addressing nucleocytoplasmic heterogeneity, the challenge of high-throughput screening remains. Although a droplet-based approach was developed for single-cell miRNA assays, the challenge of quantifying miRNA with high sensitivity to indicate nucleocytoplasmic heterogeneity remains.

Plasmonic droplet screen for single-cell secretion analysis.

Single-cell secretion analysis technologies are needed to elucidate the heterogeneity of cellular functionalities. Although ligand binding assays in microwells provide a promising approach for measuring single-cell secretions, their throughput is limited. Recently, droplet assays have been developed for high-throughput single-cell screening.

Nano-in-Micro Smart Hydrogel Composite for a Rapid Sensitive Immunoassay.

Immunoassays are an important tool in various bioanalytical settings, such as clinical diagnostics, biopharmaceutical analysis, environmental monitoring, and food testing. An enzyme-linked immunosorbent assay (ELISA) is usually used to amplify immunoassay signals; however, it requires labor-intensive and time-consuming procedures, which hinders its application to rapid cytokine detection. In this study, a nano-in-micro composite system, where immunosensing polystyrene beads (≈320 nm) are incorporated within a stimuli-responsive microgel matrix (≈40 µm) via microfluidics, is investigated.

Ultrafast Single-Cell Level Enzymatic Tumor Profiling.

In the context of tumor analysis, the implementation of precision medicine requires on-time clinical measurements, which requires rapid large-scale single-cell screening that obtains cell population distributions and functions in tumors to determine disease progression for therapeutics. In this study, a high-throughput screening (HTS) platform integrating optical fluorescence detectors and a computational method was developed as a droplet-based microfluidic flow cytometer (Droplet-μFC) to comprehensively analyze multiple proteolytic activities of a patient-derived tumor (with ∼0.5-2 M cells) at single-cell resolution within 2 h.

Smart Hydrogel Microfluidics for Single-Cell Multiplexed Secretomic Analysis with High Sensitivity.

Secreted proteins determine a range of cellular functionalities correlated with human health and disease progression. Because of cell heterogeneity, it is essential to measure low abundant protein secretions from individual cells to determine single-cell activities. In this study, an integrated platform consisting of smart hydrogel immunosensors for the sensitive detection of single-cell secretions is developed.

Interactions between bacterial surface and nanoparticles govern the performance of "chemical nose" biosensors.

Rapid and portable diagnosis of pathogenic bacteria can save lives lost from infectious diseases. Biosensors based on a "chemical nose" approach are attracting interest because they are versatile but the governing interactions between bacteria and the biosensors are poorly understood. Here, we use a "chemical nose" biosensor based on gold nanoparticles to explore the role of extracellular polymeric substances in bacteria-nanoparticle interactions.

Characteristic investigation of scanning surface plasmon microscopy for nucleotide functionalized nanoarray.

A calculation based on surface plasmon coupling condition and Maxwell-Garnett equation was performed for predicting the coupling angle shift and thin film thickness in scanning surface plasmon microscopy (SSPM). The refractive index sensitivity and lateral resolution of an SSPM system was also investigated. The limit of detection of angle shift was 0.

Tip-enhanced fluorescence with radially polarized illumination for monitoring loop-mediated isothermal amplification on hepatitis C virus cDNA.

A tip nanobiosensor for monitoring DNA replication was presented. The effects of excitation power and polarization on tip-enhanced fluorescence (TEF) were assessed with the tip immersed in fluorescein isothiocyanate solution first. The photon count rose on average fivefold with radially polarized illumination at 50 mW.

Disposable surface plasmon resonance aptasensor with membrane-based sample handling design for quantitative interferon-gamma detection.

ELISA and ELISPOT methods are utilized for interferon-gamma (IFN-γ) release assays (IGRAs) to detect the IFN-γ secreted by T lymphocytes. However, the multi-step protocols of the assays are still performed with laboratory instruments and operated by well-trained people. Here, we report a membrane-based microfluidic device integrated with a surface plasmon resonance (SPR) sensor to realize an easy-to-use and cost effective multi-step quantitative analysis.

Label-free colorimetric aptasensor for IgE using DNA pseudoknot probe.

The development of simple and low-cost approaches to the detection of immunoglobulin E (IgE) would provide a method for the early diagnosis and prevention of atopic diseases. The current methods of detection are generally tedious, multi-step processes and are limited by the high cost of the labeled proteins. We describe here a label-free structure-switching colorimetric method for the simple measurement of IgE using DNA pseudoknot probes and gold nanoparticles.

Nanodots array rapidly fabricated by Dip-Pen nanolithography with temperature and humidity control.

This study demonstrates the advantage of Dip-Pen Nanolithography (DPN) as a research and design tool for metal nano-structure fabrications. We design two different gold nano-structures, which are fabricated by DPN etching method with temperature and humidity control. The plasmon resonance frequencies of both structures are measured with dark field scattering spectroscopy.

Metallic tip enhanced fluorescence for DNA replication monitoring.

We have successfully performed localized loop-mediated isothermal reactions of hepatitis B virus (HBV) and hepatitis C virus (HCV) on the apex (50~100 nm) of metallic tips coated with Bst polymerases. The SYBR green molecules binding to the new formed HBV DNA inside the optical near fields were excited by two-photon fluorescence microscopy, and directly imaged in far field. Another reporter primer is used for HCV replication detection.

Synthesis of fluorescent gold nanodot-liposome hybrids for detection of phospholipase C and its inhibitor.

We report the synthesis of fluorescent 11-mercaptoundecanoic acid-gold nanodot-liposome (11-MUA-Au ND/Lip) hybrids by incorporation of gold nanoparticles (∼3 nm) and 11-MUA molecules in hydrophobic phospholipid membranes that self-assemble to form small unilamellar vesicles. A simple and homogeneous fluorescence assay for phospholipase C (PLC) was developed on the basis of the fluorescence quenching of 11-MUA-Au ND/Lip hybrids in aqueous solution. The fluorescence of the 11-MUA-Au ND/Lip hybrids is quenched by oxygen (O2) molecules in solution, and quenching is reduced in the presence of PLC.

Gold nanorods as probes in two-photon fluorescence correlation spectroscopy: emerging applications and potential artifacts.

Owing to the highly efficient two-photon fluorescence of gold nanorods and very short fluorescence lifetime compared with the rotational correlation time, the rotation and diffusion of a single gold nanorod can be easily observed by two-photon fluorescence correlation spectroscopy (TP-FCS). This property, along with the previous successful use as a contrast agent in two-photon fluorescence imaging, suggests a potential application in TP-FCS as well. Although the FCS measurement becomes highly efficient with gold nanorods as probes, the amplitude and temporal decay of the measured correlation functions depend critically on excitation power.

Aptamer-based colorimetric detection of platelet-derived growth factor using unmodified gold nanoparticles.

We developed a simple method for the detection of platelet-derived growth factors (PDGFs) based on base stacking effect coupled with an unmodified gold nanoparticle (AuNP) indicator. In the absence of a target, an aptamer probe and a capture probe stably co-exist in a solution, as it is difficult to sustain an interaction between both these probes due to the short 8bp duplex. However, when a target protein binds to the aptamer probe, the strong base stacking effect can lead to a favorable and stable interaction between the aptamer and capture probes.

Diagnostic devices for isothermal nucleic acid amplification.

Since the development of the polymerase chain reaction (PCR) technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling.

Detection of tip-enhanced fluorescence from loop-mediated isothermal amplification of hepatitis B virus by two-photon microscopy.

Tip-enhanced fluorescence of localized DNA replication by loop-mediated isothermal amplification (LAMP) is a potential way to observe real-time biological reaction confined in nanometer scale. We successfully coated Bst polymerase on the apex (~100 nm) of an atomic force microscope (AFM) tip and performed localized LAMP reaction of hepatitis B virus (HBV). By using this tip-based reaction, the replicated HBV DNA can be directly imaged to be 400~500 nm spots by using two-photon excitation fluorescence microscopy.

A polycarbonate based surface plasmon resonance sensing cartridge for high sensitivity HBV loop-mediated isothermal amplification.

In this study, we report a simple, low-cost surface plasmon resonance (SPR)-sensing cartridge based on a loop-mediated isothermal amplification (LAMP) method for the on-site detection of the hepatitis B virus (HBV). For LAMP detection, a SPR based LAMP sensing system (SPRLAMP) was constructed, including a novel SPRLAMP sensing cartridge integrating a polymethyl methacrylate (PMMA) micro-reactor with a polycarbonate (PC)-based prism coated with a 50 nm Au film. First, we found that the change of refractive index of the bulk solution was approximately 0.

An amplified surface plasmon resonance "turn-on" sensor for mercury ion using gold nanoparticles.

Inorganic mercury ion (Hg(2+)) has been shown to coordinate to DNA duplexes that feature thymine-thymine (T-T) base pair mismatches. This observation suggests that an Hg(2+)-induced conformational change in a single-stranded DNA molecule can be used to detect aqueous Hg(2+). Here, we have developed an analytical method using surface plasmon resonance (SPR) to develop a highly selective and sensitive detection technique for Hg(2+) that takes advantage of T-Hg(2+)-T coordination chemistry.