Wei Jin

Integrated Glass Microdevice for Nucleic Acid Purification, Loop-Mediated Isothermal Amplification, and Online Detection

A microdevice made of glass for genetic analysis has been fabricated, for the first time, for integration of extraction of nucleic acids and loop-mediated isothermal amplification (LAMP), followed by online fluorescence detection of amplification products on a single chip. The nucleic acid (NA) extraction region consists of a microfabricated serpentine channel in which micropillars were etched to increase the channel surface area and the capture efficiency of NAs. Nucleic acid molecules were bound to these pillars and channel surface in the presence of the chaotropic salt guanidine hydrochloride and eluted into a downstream amplification chamber with low ionic strength buffer where loop-mediated isothermal amplification was efficiently performed. Amplification can be detected online by the increase of fluorescence intensity at 540 nm when a low concentration of SYBR Green I, a fluorescent dsDNA intercalating dye, is employed. Flow control was accomplished by using laminar flow and differential channel flow resistances. Through passivation of the LAMP chamber and the channel between the extraction region and amplification domain, effective nucleic acid extraction and amplification were performed by just using a double-channel syringe pump and a heating block. By using this integrated microdevice, the purification of nucleic acids from complex biological matrixes and their subsequent amplification and detection online could be finished within 2 h.

A DNA sensor based on surface plasmon resonance for apoptosis-associated genes detection.

A practical and simple DNA sensor based on surface plasmon resonance (SPR) had been developed to determine apoptosis-associated genes, bcl-2 and bax. This SPR sensor was designed on the basis of simultaneous multi-wavelength detection. The complementary sequences of bcl-2 or bax oligonucleotide labeled with biotin were used as the probes. Biotin-avidin system was used to immobilize the bio-DNA on the sensor surface. The assembling processes and conditions for the DNA sensor were examined. The SPR sensor could be used to monitor the process of the immobility of the bio-DNA and DNA hybridization in real-time. The determination range of bcl-2 and bax oligonucleotide (20 bases) were 50-400 ng/mL. The determination range of polymerase chain reaction (PCR) product of bcl-2 (405 bases) was 5-60 ng/mL and PCR product of bax (538 bases) was 5-40 ng/mL. The stability, reversibility and specificity of the DNA sensor were also investigated. It was found from the experiment that the sensor could be applied for a quite long time (about 90 times). The relative standard deviation (R.S.D.) for determination oligonucleotides and PCR products of bcl-2 were 1.2 and 1.3%, respectively. The interference of noncomplementary DNA sequence with the determination of DNA was examined and it was found that noncomplementary 20-mer and 21-mer DNA (p53 and p21) do not affect the determination of bcl-2 or bax. This device could be used to study apoptosis and signal transduction routine genes. The sensor was shown to be of simplicity, sensitivity, selectivity, rapid response and cost effectiveness.

Au:CdHgTe quantum dots for in vivo tumor-targeted multispectral fluorescence imaging

AbstractNear-infrared gold-doped CdHgTe quantum dots (QDs) with improved photoluminescence and biocompatibility were developed using an aqueous solution route with l-glutathione and l-cysteine as stabilizers. As-prepared Au:CdHgTe QDs were covalently linked to arginine–glycine–aspartic acid (RGD) peptide, anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MAb), and anti- carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) MAb separately. Three Au:CdHgTe QD bioconjugates (QD800-RGD, QD820-anti-CEACAM1, and QD840-anti-EGFR) were successfully used as probes for in vivo tumor-targeted multispectral fluorescence imaging of xenografts. Fluorescence signals from the QD bioconjugates used to detect three tumor markers were spectrally unmixed, and their co-localization was analyzed. The results indicate that multiple tumor markers could be simultaneously detected by multispectral fluorescence imaging in vivo using QD bioconjugates as probes. This approach has excellent potential as an imaging method for the noninvasive exploration and detection of multiple tumor markers in vivo, thereby substantially aiding the diagnosis of cancer.n FigureIn vivo tumor-targeted multispectral fluorescence imaging with Au:CdHgTe quantum dots

Mixed-Dye-Based Label-Free and Sensitive Dual Fluorescence for the Product Detection of Nucleic Acid Isothermal Multiple-Self-Matching-Initiated Amplification.

Visual detections based on fluorescence and the color changes under natural light are two promising product detections for isothermal nucleic acid amplifications (INAAs) such as the isothermal multiple-self-matching-initiated amplification (IMSA) as point-of-care testing techniques. However, the currently used approaches have shortcomings in application. For the former, fluorescence changes recognized by naked eye may be indistinguishable because of single fluorescence emitted and strong background noise, which requires empirical preset of cutoff intensity values. For the latter, visual detection sensitivity under natural light is not comparable to that based on fluorescence. Herein, hydroxyl naphthol blue (HNB) and SYBR Green I (SG) were coupled to acquire a label-free dual fluorescence for the visual product detection of IMSA. The mixed-dye-loaded off-chip (tube-based) and on-chip (microfluidic chip-based) IMSAs for the detection of hepatitis B virus were conducted. The results demonstrated that this dual fluorescence could realize distinguishable fluorescent color changes to improve visual detection sensitivity and avoid the preset of cutoff values. Moreover, the mixed dye is stable when kept at room temperature and compatible with the IMSAs reagents without a contamination-prone step of opening tubes after amplification. Also, this coupled dye inherits the advantages of achieving color changes under natural light from HNB and real-time detection from SG. In conclusion, the mixed-dye-based dual fluorescence has a potential in the point-of-care testing application for realizing off-chip and on-chip product detection of IMSA, loop-mediated isothermal amplification (LAMP), or other INAAs.

Detection of mismatched caspase-3 DNA oligonucleotides with an SPR biosensor following amplification by Taq polymerase

AbstractWe demonstrate that base mismatches of caspase-3 DNA sequences can be detected by surface plasmon resonance (SPR) following signal amplification by polymerase from Thermus aquaticus (Taq). The concentration of magnesium ions and the respective dNTPs for polymerase binding to the oligonucleotides on the sensing surface were optimized. Taq polymerase binds to double-stranded DNA that is self-assembled on the gold surface of the biosensor to induce an SPR signal. Experiments are presented on the effect of Mg(II) and dNTP concentrations on the activity of the polymerase on the sensing surface. The detection limits are 50xa0pM, 0.1xa0nM, 0.7xa0nM, 7xa0nM, and 20xa0nM for correctly matched, single-base mismatched, two-base mismatched, three-base mismatched and four-base mismatched DNA of caspase-3, respectively. This is attributed to the optimized experimental conditions, with samples containing 2xa0μM of Mg(II) and 0.3xa0mM of dNTP.n FigureThe process of detecting mismatched caspase-3 DNA oligonucleotides with SPR biosensor

Design and implementation of an in vivo 3-D fluorescent imaging system

Fluorescence has been proved to be a versatile and useful tool for animal in vivo imaging. It has been used widely in biomedical research and drug discoveries. With the recent development of the technology and instruments, the 2-D imaging system has been developed to a 3-D imaging in vivo. 3-D imaging will provide researchers detailed spatial information of the targets and its relationship with animal organs. This paper introduces a new design of an in vivo 3-D multispectral fluorescent imaging system, including the design of hardware platform and the imaging algorithm based on binocular stereo vision theory. The working principle of the system, structure of the hardware and the system diagram are introduced. The algorithm of binocular stereo vision is utilized to complete camera calibration, stereo rectification, and 3D coordinates computing based on OpenCV.

Recovering high dynamic range radiance maps from in vivo fluorescence molecular imaging

A recent development in molecular imaging is in vivo fluorescence molecular imaging with near-infrared light, which has extraordinary significance in early diagnosis of the disease. This paper presents a method of recovering high dynamic range radiance maps at in vivo fluorescence molecular imaging technology. Use differently exposed photographs to recover the response curve of the imaging process, with the known curve, can fuse a single, high dynamic range radiance map whose pixel values are proportional to the true fluorescence signal values in the scene. Will contribute to quilitative and quatitative analysis, offer better observation, and get information much more accurate and true. Have great significance for analysis and diagnosis.

An Optical Immunosensor Based on Surface Plasmon Resonance for Human cTnI Determination

Human cardiac troponin I (cTnl) is a specific protein of cardiac muscle. The detection of the content of cTnl in human serum shows high specificity for diagnosing acute myocardial infarction. For preparation of diagnostic reagents of myocardial infarction, cTnl is isolated directly from human cardiac muscle by skeletal TnC-sepharose 4B affinity chromatographic column. The molecular weight and the amino acid composition of isolated cTnl is consistent with reference. A novel optical biosensor based on simultaneous multiwave-length detected surface plasmon resonance (SPR) has been developed for monitoring cTnl. The sensor is designed on the basis of fixing angle of incidence and measuring the reflected intensities in the wavelength range of 400-800 nm. The SPR spectra are shown in terms of reflected light intensity verus wavelength of incident light. The intensity of the reflected light is the minimum at the resonant wavelength. Molecular self-assembling in solution is used to form the sensing membrane on the gold substrate. The processes of sensing monolayer formation were studied in real time through observing the change of resonant wavelength. The modified surface was used as a model immunosensor and successfully detected cTnl. cTnC was detected in the concentration range from 0.143 ~ 50 mug/mL. Under optimum experimental conditions, the sensor has a good repeatability, reversibility and selectivity.