Jinzhao Song

Aptamer-based polymerase chain reaction for ultrasensitive cell detection.

A new system was developed for sensitive and selective detection of tumor cells taking advantage of cell-attached aptamers amplified by PCR and output signals amplified by cationic conjugated polymers.

Multicellular Assembly and Light-Regulation of Cell–Cell Communication by Conjugated Polymer Materials

Using cell-surface modification and biotin-streptavidin interactions, immune cells and target tumor cells are made to form multicellular assemblies. A polythiophene derivative can undergo cellular uptake, allowing the sensitization of oxygen under light irradiation. The subsequent generation of reactive oxygen species (ROS) regulates cell-cell communication in time and space.

Multiplex Detection of DNA Mutations by the Fluorescence Fingerprint Spectrum Technique

A fingerprint spectrum technique that utilizes cationic conjugated-polymer-based fluorescence resonance energy transfer (FRET) is used for multiplex detection of DNA mutations. This method detects as low as 5 % mutation of the total DNA. Ten PIK3CA mutations originating from 30 clinical breast cancer samples could be detected.

Visual detection of DNA mutation using multicolor fluorescent coding.

A visual colorimetric method for detecting multiplex DNA mutations has been developed using multicolor fluorescent coding based on multistep FRET of cationic conjugated polymers. Expensive instruments and technical expertise are not required in this method. Also our visual system provides a quantitative detection by simply analyzing RGB values of images. Genomic DNAs extracted from 60 FFPE colon tissues can be sensitively determined by utilizing our visual assay with a high-throughput manner. Thus, it proves to be sensitive, reliable, cost-effective, simple, and high-throughput for mutation detection.

Two-Stage Isothermal Enzymatic Amplification for Concurrent Multiplex Molecular Detection

BACKGROUND The wide array of pathogens responsible for infectious diseases makes it difficult to identify causative pathogens with single-plex tests. Although multiplex PCR detects multiple targets, it is restricted to centralized laboratories, which delays test results or makes multiplexing unavailable, depriving healthcare providers of critical, real-time information. METHODS To address the need for point-of-care (POC) highly multiplexed tests, we propose the 2-stage, nested-like, rapid (<40 min) isothermal amplification assay, dubbed rapid amplification (RAMP). RAMPs first-stage uses outer loop-mediated isothermal amplification (LAMP) primers to amplify all targets with recombinase polymerase amplification (RPA). First-stage amplicons are aliquoted to second stage reactors, each specialized for a specific target, to undergo LAMP. The assay is implemented in a microfluidic chip. LAMP amplicons are detected in situ with colorimetric dye or with a fluorescent dye and a smartphone. RESULTS In experiments on a benchtop and in a microfluidic format, RAMP demonstrated high level of multiplexing (≥16); high sensitivity (i.e., 1 plaque-forming unit of Zika virus) and specificity (no false positives or negatives); speed (<40 min); ease of use; and ability to cope with minimally processed samples. CONCLUSIONS RAMP is a hybrid, 2-stage, rapid, and highly sensitive and specific assay with extensive multiplexing capabilities, combining the advantages of RPA and LAMP, while circumventing their respective shortcomings. RAMP can be used in the lab, but one of its distinct advantages is amenability to simple implementation in a microfluidic format for use at the POC, providing healthcare personnel with an inexpensive, highly sensitive tool to detect multiple pathogens in a single sample, on site.

Molecular Detection of Schistosome Infections with a Disposable Microfluidic Cassette.

Parasitic helminths such as schistosomes, as well as filarial and soil-transmitted nematodes, are estimated to infect at least a billion people worldwide, with devastating impacts on human health and economic development. Diagnosis and monitoring of infection dynamics and efficacy of treatment depend almost entirely on methods that are inaccurate, labor-intensive, and unreliable. These shortcomings are amplified and take on added significance in mass drug administration programs, where measures of effectiveness depend on accurate monitoring of treatment success (or failure), changes in disease transmission rates, and emergence of possible drug resistance. Here, we adapt isothermal molecular assays such as loop-mediated isothermal amplification (LAMP) to a simple, hand-held, custom-made field-ready microfluidic device that allows sensitive and specific detection of schistosome cell-free nucleic acids in serum and plasma (separated with a point-of-care plasma separator) from Schistosoma mansoni-infected mice. Cell-free S. mansoni DNA was detected with our device without prior extraction from blood. Our chip exhibits high sensitivity (~2x10−17 g/μL), with a positive signal for S. mansoni DNA detectable as early as one week post infection, several weeks before parasite egg production commences. These results indicate that incorporation of isothermal amplification strategies with our chips could represent a strategy for rapid, simple, low-cost diagnosis of both pre-patent and chronic schistosome infections as well as potential monitoring of treatment efficacy.

Preparation and optical property of new fluorescent nanoparticles.

A new fluorescent nanoparticle (PIOT-HA) is synthesized with cationic polyester (PIOT) and anionic hyaluronic acid (HA) by electrostatic interactions in an aqueous solution. The nanoparticles (NPs) are degradable upon treatments with alkali or hyaluronidase, which exhibits better biological safety and potential application in vitro and in vivo. Through specific interactions between the HA locating on the surfaces of PIOT-HA NPs and the CD44 protein over-expressed on the MDA-MB-231 cancer cell line, PIOT-HA NPs could selectively image the cancer cells. Upon white light irradiation, the PIOT-HA NPs can sensitize oxygen to generate reactive oxygen species (ROS) that inactivate the neighboring CD44 protein, which inhibits the migration of MDA-MB-231 cancer cells.

A Reverse Transcription Loop-Mediated Isothermal Amplification Assay Optimized to Detect Multiple HIV Subtypes

Diagnostic methods for detecting and quantifying HIV RNA have been improving, but efficient methods for point-of-care analysis are still needed, particularly for applications in resource-limited settings. Detection based on reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is particularly useful for this, because when combined with fluorescence-based DNA detection, RT-LAMP can be implemented with minimal equipment and expense. Assays have been developed to detect HIV RNA with RT-LAMP, but existing methods detect only a limited subset of HIV subtypes. Here we report a bioinformatic study to develop optimized primers, followed by empirical testing of 44 new primer designs. One primer set (ACeIN-26), targeting the HIV integrase coding region, consistently detected subtypes A, B, C, D, and G. The assay was sensitive to at least 5000 copies per reaction for subtypes A, B, C, D, and G, with Z-factors of above 0.69 (detection of the minor subtype F was found to be unreliable). There are already rapid and efficient assays available for detecting HIV infection in a binary yes/no format, but the rapid RT-LAMP assay described here has additional uses, including 1) tracking response to medication by comparing longitudinal values for a subject, 2) detecting of infection in neonates unimpeded by the presence of maternal antibody, and 3) detecting infection prior to seroconversion.

Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification

Microfluidic components and systems for rapid (<60 min), low-cost, convenient, field-deployable sequence-specific nucleic acid-based amplification tests (NAATs) are described. A microfluidic point-of-care (POC) diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of “lab on a chip” NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (polymerase-chain reaction)-based tests in clinical laboratories can be realized with a disposable, palm-sized, plastic microfluidic chip in which: (1) nucleic acids (NAs) are extracted from relatively large (~mL) volume sample lysates using an embedded porous silica glass fiber or cellulose binding phase (“membrane”) to capture sample NAs in a flow-through, filtration mode; (2) NAs captured on the membrane are isothermally (~65 °C) amplified; (3) amplicon production is monitored by real-time fluorescence detection, such as with a smartphone CCD camera serving as a low-cost detector; and (4) paraffin-encapsulated, lyophilized reagents for temperature-activated release are pre-stored in the chip. Limits of Detection (LOD) better than 103 virons/sample can be achieved. A modified chip with conduits hosting a diffusion-mode amplification process provides a simple visual indicator to readily quantify sample NA template. In addition, a companion microfluidic device for extracting plasma from whole blood without a centrifuge, generating cell-free plasma for chip-based molecular diagnostics, is described. Extensions to a myriad of related applications including, for example, food testing, cancer screening, and insect genotyping are briefly surveyed.

Fully 3D printed integrated reactor array for point-of-care molecular diagnostics.

Molecular diagnostics that involve nucleic acid amplification tests (NAATs) are crucial for prevention and treatment of infectious diseases. In this study, we developed a simple, inexpensive, disposable, fully 3D printed microfluidic reactor array that is capable of carrying out extraction, concentration and isothermal amplification of nucleic acids in variety of body fluids. The method allows rapid molecular diagnostic tests for infectious diseases at point of care. A simple leak-proof polymerization strategy was developed to integrate flow-through nucleic acid isolation membranes into microfluidic devices, yielding a multifunctional diagnostic platform. Static coating technology was adopted to improve the biocompatibility of our 3D printed device. We demonstrated the suitability of our device for both end-point colorimetric qualitative detection and real-time fluorescence quantitative detection. We applied our diagnostic device to detection of Plasmodium falciparum in plasma samples and Neisseria meningitides in cerebrospinal fluid (CSF) samples by loop-mediated, isothermal amplification (LAMP) within 50 min. The detection limits were 100 fg for P. falciparum and 50 colony-forming unit (CFU) for N. meningitidis per reaction, which are comparable to that of benchtop instruments. This rapid and inexpensive 3D printed device has great potential for point-of-care molecular diagnosis of infectious disease in resource-limited settings.