Zhike He

Simultaneous Determination of Human Enterovirus 71 and Coxsackievirus B3 by Dual-Color Quantum Dots and Homogeneous Immunoassay

Human Enterovirus 71 (EV71) and Coxsackievirus B3 (CVB3) have high risks for morbidity and mortality. A virus quantitation immunoassay has been proposed by employing two colored quantum dots (QDs), antibodies of the virus, and graphene oxide (GO). The QDs are streptavidin-conjugated quantum dots (SA-QDs), and the antibodies are biotinylated antibodies. Biotinylated EV71 antibody (Ab1) was associated with 525 nm green colored SA-QDs via biotin-streptavidin interaction forming QDs-Ab1, whereas biotinylated CVB3 antibody (Ab2) was associated with 605 nm red colored SA-QDs via biotin-streptavidin interaction forming QDs-Ab2. GO was an excellent quencher to the fluorescence of both QDs-Ab1 and QDs-Ab2. The targets of EV71 and CVB3 can break up the complex of QDs-Ab and GO, recovering the fluorescence of QDs-Ab1 and QDs-Ab2, respectively. Using these two different colored QDs-Ab fluorescence recovery intensities upon the addition of targets EV71 and CVB3, the two enteroviruses can be simultaneously quantitatively determined with a single excitation light. The detection limits of EV71 and CVB3 are 0.42 and 0.39 ng mL(-1) based on 3 times signal-to-noise ratio, respectively. More importantly, this strategy can be further used as a universal method for any protein or virus determination by changing the conjugated antibodies in disease early diagnosis, which can provide a fast and promising clinical approach for virus differentiation and determination. In a word, a simple, fast, sensitive, and highly selective assay for EV71 and CVB3 has been developed. It could be applied in clinical sample analysis with a satisfactory result. It was notable that the sensor could not only achieve rapid and precise quantitative determination of protein/virus by fluorescent intensity but also could be applied in semiquantitative protein/virus determination by digital visualization.

Determination of glucose and uric acid with bienzyme colorimetry on microfluidic paper-based analysis devices

In this work, we first employ a drying method combining with the bienzyme colorimetric detection of glucose and uric acid on microfluidic paper-based analysis devices (μPADs). The channels of 3D μPADs are also designed by us to get better results. The color results are recorded by both Gel Documentation systems and a common camera. By using Gel Documentation systems, the limits of detection (LOD) of glucose and uric acid are 3.81 × 10(-5)M and 4.31 × 10(-5)M, respectively one order of magnitude lower than that of the reported methods on μPADs. By using a common camera, the limits of detection (LOD) of glucose and uric acid are 2.13 × 10(-4)M and 2.87 × 10(-4)M, respectively. Furthermore, the effects of detection conditions have been investigated and discussed comprehensively. Human serum samples are detected with satisfactory results, which are comparable with the clinical testing results. A low-cost, simple and rapid colorimetric method for the simultaneous detection of glucose and uric acid on the μPADs has been developed with enhanced sensitivity.

On-demand preparation of quantum dot-encoded microparticles using a droplet microfluidic system

Optical barcoding technology based on quantum dot (QD)-encoded microparticles has attracted increasing attention in high-throughput multiplexed biological assays, which is realized by embedding different-sized QDs into polymeric matrixes at precisely controlled ratios. Considering the advantage of droplet-based microfluidics, producing monodisperse particles with precise control over the size, shape and composition, we present a proof-of-concept approach for on-demand preparation of QD-encoded microparticles based on this versatile new strategy. Combining a flow-focusing microchannel with a double T-junction in a microfluidic chip, biocompatible QD-doped microparticles were constructed by shearing sodium alginate solution into microdroplets and on-chip gelating these droplets into a hydrogel matrix to encapsulate CdSe/ZnS QDs. Size-controllable QD-doped hydrogel microparticles were produced under the optimum flow conditions, and their fluorescent properties were investigated. A novel multiplex optical encoding strategy was realized by loading different sized QDs into a single droplet (and thus a hydrogel microparticle) with different concentrations, which was triggered by tuning the flow rates of the sodium alginate solutions entrapped with different-colored QDs. A series of QD-encoded microparticles were controllably, and continuously, produced in a single step with the present approach. Their application in a model immunoassay demonstrated the potential practicability of QD-encoded hydrogel microparticles in multiplexed biomolecular detection. This simple and robust strategy should be further improved and practically used in making barcode microparticles with various polymer matrixes.

Synthesis and characterization of high-quality water-soluble CdTe: Zn2+ quantum dots capped by N-acetyl-L-cysteineviahydrothermal method

Novel, water-soluble Zn2+ doped CdTe quantum dots (QDs) were prepared through a one-step hydrothermal route. Due to high temperature and high pressure in the hydrothermal route, the addition of Zn ions and employment of a new stabilizer N-acetyl-L-cysteine (NAC), the as-prepared CdTe: Zn2+ QDs (emission wavelength at 530–623 nm) exhibit a high quantum yield (up to 75.31%), high doping ratio (the actual constituent ratio reaches 2.41u2006:u20061.00), high stability and excellent biocompitability. The characterization of as-prepared QDs was carried out through fluorescence spectroscopy, UV absorption spectroscopy, Fourier transform infrared spectroscopy and transmission electron microscopy. In particular, for the first time, we realized qualitative, semi-quantitative and quantitative studies on the doping of Zn to CdTe QDs through X-ray photoelectron spectroscopy, electron diffraction spectroscopy and atomic absorption spectrometry. In addition, the relation between the feed ratio of Zn to Cd and the actual constituent ratio of the prepared QDs was also examined.

One‐Pot Synthesized DNA–CdTe Quantum Dots Applied in a Biosensor for the Detection of Sequence‐Specific Oligonucleotides

In the past decade, as a new class of fluorescent probes, semiconductor quantum dots (QDs) have been widely applied in the area of biosensing, immunoassays, and biological imaging because of their unique optical characteristics and biocompatibility. In these applications, a number of biomolecule-labeled QDs were devoted to biospecific interactions, such as DNA-labeled QDs. These QDs can be usually used as a donor for molecular recognition mainly in fluorescence resonance energy transfer (FRET). The coupling reaction between streptavidin QDs and biotin–DNA is a common approach for DNA-labeled QDs for the streptavidin–biotin specific reaction. The method is convenient, but it has no advantage in FRET application, due to increasing the distance between the donor (the large size of the QD) and the acceptor. Furthermore, streptavidin QDs are expensive. DNA covalent bonding to QDs by reaction between the carboxy/amino group of QDs and amino/carboxy group modified DNA could overcome these flaws; however; the acidic coupling conditions may lead to the breakdown of the QDs, which limits its application. Zhou et al. directly coupled a dye-labeled DNA acceptor to a QD donor through a thiol linker. This method costs less, significantly reduces the donor–acceptor distance to increase the FRET efficiency, and at the same time retains the stability of the QDs . However, the process is multistep and complicated. A one-step synthesis of DNA-labeled QDs has been established in such an environment. Phosphorothiolate phosphate (ps–po) DNA compounds were used as ligands to obtain DNA-labeled QDs. Herein, we have synthesized nucleic acid functionalized CdTe nanocrystals with different emission wavelengths by a one-step synthesis. Recently, new emerging zero-bandgap carbon nanomaterials, graphene, and graphene oxide (GO) have attracted great interest in the fields of biology, chemistry, physics, and materials. Graphene and GO have p-systems, which allows them very easily to accept electrons. Inspired by the property, researchers have used graphene and GO as energy acceptors to develop many biosensors based on the FRET system. Importantly, graphene and GO possess a superquenching ability that is critical for the sensitivity of assays. In addition, graphene displays better conductivity than GO, because GO has many oxygen-containing groups, which destroy the conjugate structure. Thus, graphene was chosen as the energy acceptor in this paper. A biosensor, which is based on FRET from the DNA-functionalized CdTe nanocrystals to graphene, has been developed, that is, DNA–CdTe QDs prepared by a one-pot method, and its successful application in the analytical detection of the hepatitis B virus (HBV) surface–antigen gene is described for the first time. Scheme 1 shows the preparation of DNA–CdTe QDs by a one-pot method. Glutathione (GSH) and a specific-sequence DNA were used as a co-ligands to stabilize the QDs. The DNA ligand consisted of two domains: phosphorothio-

Encapsulating Quantum Dots into Enveloped Virus in Living Cells for Tracking Virus Infection

Utilization of quantum dots (QDs) for single virus tracking has attracted growing interest. Through modification of viral surface proteins, viruses can be labeled with various functionalized QDs and used for tracking the routes of viral infections. However, incorporation of QDs on the viral surface may affect the efficiency of viral entry and alter virus-cell interactions. Here, we describe that QDs can be encapsulated into the capsid of vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentivirus (PTLV) in living cells without modification of the viral surface. QDs conjugated with modified genomic RNAs (gRNAs), which contain a packaging signal (Psi) sequence for viral genome encapsulation, can be packaged into virions together with the gRNAs. QD-containing PTLV demonstrated similar entry efficiency as the wild-type PTLV. After infection, QD signals entered the Rab5+ endosome and then moved to the microtubule organizing center of the infected cells in a microtubule-dependent manner. Findings in this study are consistent with previously reported infection routes of VSV and VSV-G pseudotyped lentivirus, indicating that our established QD packaging approach can be used for enveloped virus labeling and tracking.

Magnetic microparticle-based multiplexed DNA detection with biobarcoded quantum dot probes

We have developed a new analytical method to detect multiple DNA simultaneously based on the biobarcoded CdSe/ZnS quantum dot (QD) and magnetic microparticle (MMP). It was demonstrated by using oligonucleotide sequences of 64 bases associated with human papillomavirus 16 and 18 L1 genes (HPV-16 and HPV-18) as model systems. This analytical system involves three types of probes, a MMP probe and two streptavidin-modified QD probes. The MMPs are functionalized with HPV-16 and HPV-18 captures DNA to form MMP probes. The QDs are conjugated with HPV-16 or HPV-18 probe DNA along with FAM- or Rox-labeled random DNA to form HPV-16 and HPV-18 QD probes, respectively. A one-step hybridization reaction was performed by mixing the MMP probes, HPV-16 and HPV-18 target DNA (T-16 and T-18), HPV-16 and HPV-18 QD probes. Afterwards, the hybrid-conjugated microparticles were separated by a magnet and heated to remove the MMPs. Finally, the detections of T-16 and T-18 were done by measuring fluorescence signals of FAM and Rox, respectively. Under the optimum conditions, the fluorescence intensity exhibited a good linear dependence on target DNA concentration in the range from 8 × 10⁻¹¹ to 8 × 10⁻⁹ M. The detection limit of T-16 is up to 7 × 10⁻¹¹ M (3σ), and that of T-18 is 6 × 10⁻¹¹ M. Compared with other biobarcode assay methods, the proposed method that QDs were used as the solid support has some advantages including shorter preparation time of QD probes, faster binding kinetics and shorter analytical time. Besides, it is simple and accurate.

Dual-color fluorescence and homogeneous immunoassay for the determination of human enterovirus 71.

We have developed a new fluorescent immune ensemble probe comprised of a conjugated lower toxic water-soluble CdTe:Zn(2+) quantum dots (QDs) and Ru(bpy)(2)(mcbpy-O-Su-ester)(PF(6))(2)- antibody complex (Ru-Ab) for the dual-color determination of human enterovirus 71 (EV71) in homogeneous solution. EV71 monoantibody was easily covalently conjugated with Ru(bpy)(2)(mcbpy-O-Su-ester)(PF(6))(2) to form a stable complex Ru-Ab, which acted both as an effective quencher of QDs fluorescence and the capture probe of virus antigen EV71. Herein, the target EV71 can break up the low fluorescent ionic ensemble by antigen-antibody combination to set free the fluorescent QDs and restore the fluorescence of QDs whereas the fluorescence intensity of Ru-Ab remains the same. Thus, the determination of EV71 by the complex Ru-Ab and QDs can be realized via the restoration of QDs fluorescence upon addition of EV71 and even can be directly evaluated by the ratio of green-colored QDs fluorescence intensity to Ru-Ab red-colored fluorescence intensity. The green-colored fluorescence of QDs was very sensitive to the change of EV71 concentration, and its fluorescence intensity increased with the increase of EV71 concentration between 1.8 ng/mL and 12 μg/mL. With this method, EV71 was detected at subnanogram per milliliter concentration in the presence of 160 μg/mL bovine serum albumin. More importantly, this strategy can be used as a universal method for any protein or virus by changing conjugated antibodies in disease early diagnosis providing a fast and promising clinical approach for virus determination. In a word, a simple, fast, sensitive, and highly selective assay for EV71 has been described. It could be applied in real sample analysis with a satisfactory result. It was notable that the sensor could not only achieve rapid and precise quantitative determination of protein/virus by fluorescent intensity but also could be applied in semiquantitative protein/virus determination by digital visualization.

Real-time luminescence-based colorimetric determination of double-strand DNA in droplet on demand

We have developed a new luminescence-based colorimetric droplet platform for the determination of double-stranded DNAs (dsDNA). This colorimetric sensor was realized via choosing a fluorescent ensemble probe comprising water-soluble N-acetylcysteine-capped CdTe quantum dots (QDs) and Ru(bpy)(2)(dppx)(2+) (Ru). To provide a convenient and low cost droplet platform for colorimetry, the microvalve technique was adapted to adjust droplet size precisely, achieve the desired fusion of multiple droplets and trap droplets on demand, as well as implement concentration gradients of DNA on a single chip. In the colorimetric sensor, Ru served as both an effective quencher for QDs and a reporter for dsDNA. With increasing concentration of dsDNA, a gradually enhanced color response was observed because of the competition of dsDNA with QDs for Ru. Under the optimum conditions, this biosensing system exhibited not only good sensitivity and specificity for calf thymus DNA with the detection limit of 1.0 pg, but also coincident performances in diluted human serum with the detection limit of 0.9 pg. The droplet biosensor provides a highly efficient, rapid and visual method for dsDNA analysis. The colorimetric droplet platform could be useful as a simple research tool for the study of limited and precious regents such as protein and virus samples, etc.

Droplet-based microscale colorimetric biosensor for multiplexed DNA analysis via a graphene nanoprobe

The development of simple and inexpensive DNA detection strategy is very significant for droplet-based microfluidic system. Here, a droplet-based biosensor for multiplexed DNA analysis is developed with a common imaging device by using fluorescence-based colorimetric method and a graphene nanoprobe. With the aid of droplet manipulation technique, droplet size adjustment, droplet fusion and droplet trap are realized accurately and precisely. Due to the high quenching efficiency of graphene oxide (GO), in the absence of target DNAs, the droplet containing two single-stranded DNA probes and GO shows dark color, in which the DNA probes are labeled carboxy fluorescein (FAM) and 6-carboxy-X-rhodamine (ROX), respectively. The droplet changes from dark to bright color when the DNA probes form double helix with the specific target DNAs leading to the dyes far away from GO. This colorimetric droplet biosensor exhibits a quantitative capability for simultaneous detection of two different target DNAs with the detection limits of 9.46 and 9.67×10(-8)M, respectively. It is also demonstrated that this biosensor platform can become a promising detection tool in high throughput applications with low consumption of reagents. Moreover, the incorporation of graphene nanoprobe and droplet technique can drive the biosensor field one more step to some extent.