Target-induced DNA nanomachine operation for the detection of proteins.

Accurate and sensitive detection of disease-associated proteins in early stage of patients plays an important role in timely treatment and successfully extending patients' lives. To meet this demand, we herein rationally designed a flexible target-induced DNA nanomachine operation (TIDNMO) sensor for the detection of proteins. The TIDNMO system was composed of DNA nanoswitch and DNA walker. Triplex DNA nanoswitch was triggered by specific target, followed by the release of the walking strand, which initiated the DNA walker amplification as signal output. In addition, the Exo III could drive walking strand autonomously move on gold nanoparticle surface to realize 2 orders of magnitude signal amplification. What's more, this sensor could transform its suitable functional recognition element of DNA nanoswitch to recognize other specific molecule and realize different targets sensing based on identical walking tracks. Considering the facile reporter elements and efficient amplification performance, the present DNA nanomachine as a sensor could achieve a detection limit of 68 pM for anti-Dig antibody, 0.95 pM for mucin-1 respectively, along with a superb specificity. Furthermore, the method reported here opened a new chapter in disease-related protein sensing for the development of clinical early diagnosis.

A Facile Strategy for Multiplex Protein Detection by a Fluorescent Microsphere-Based Digital Immunoassay.

The digital immunoassay is a highly sensitive detection technique based on single-molecule counting and is widely used in the ultrasensitive detection of biomarkers. Herein, we developed a fluorescent microsphere-based digital immunoassay (FMDIA) by employing fluorescent microspheres as both the carriers for immunoreaction and fluorescent reports for imaging. In this approach, the target protein in the sample was captured by fluorescent microspheres to form a biotin-labeled sandwich immunocomplex, and then, the fluorescent microspheres containing the target protein molecules were captured by adding streptavidin-coated magnetic beads (SA-MBs). By counting the proportion of fluorescence-positive magnetic beads, the concentration of the target protein can be precisely quantified. As a proof of concept, α fetoprotein (AFP) and human interleukin-6 (IL-6) were used to assess the analytical performance of the proposed FMDIA, and limit of detection (LOD) values of 21 pg/mL (0.30 pM) and 0.19 pg/mL (7.3 fM) were achieved, respectively. The results of AFP detection in serum samples of patients and healthy people were consistent with the reference values given by the hospital. Furthermore, by adding fluorescent microspheres of various colors for encoding, the proposed FMDIA can easily realize the simultaneous detection of multiple proteins without the need to introduce multiple modified magnetic beads. This multiplex protein detection strategy, in which the reactions are first carried out on the fluorescent microspheres and then magnetic beads are used to capture the fluorescent reporters containing the target molecules, provides a new idea for digital assays.

Nucleic Acid Amplification-Free Digital Detection Method for SARS-CoV-2 RNA Based on Droplet Microfluidics and CRISPR-Cas13a.

Most of the methods currently developed for RNA detection based on CRISPR were combined with nucleic acid amplification. As a result, such methods inevitably led to certain disadvantages such as multiple operations, expensive reagents, and amplification bias. To solve the above problems, we developed a highly sensitive and specific nucleic acid amplification-free digital detection method for SARS-CoV-2 RNA based on droplet microfluidics and CRISPR-Cas13a. In this assay, thousands of monodisperse droplets with a size of 30 µm were generated within 2 min by a negative pressure-driven microfluidic chip. By confining a single target RNA recognition event to an independent droplet, the collateral cleavage products of activated Cas13a could be accumulated in one droplet. By combining the droplet microfluidics and CRISPR-Cas13a, SARS-CoV-2 RNA could be easily detected within 30 min with a detection limit of 470 aM. The performance of this assay was verified by specificity experiments and spiking and recovery experiments with human saliva. Compared with many developed methods for SARS-CoV-2 RNA detection, our method is time- and reagent-saving and easy to operate. Taken together, this digital detection method based on droplet microfluidics and CRISPR-Cas13a provides a promising approach for RNA detection in clinical diagnostics.

DNAzyme Walker for Homogeneous Detection of Enterovirus EV71 and CVB3.

When dealing with infectious pathogens, the risk of contamination or infection in the process of detecting them is nonnegligible. Separation-free detection will be beneficial in operation and safety. In this work, we proposed a DNAzyme walker for homogeneous and isothermal detection of enterovirus. The DNAzyme is divided into two inactivate subunits. When the subunit-conjugated antibody binds to the target virus, the activity of the DNAzyme recovers as a result of spatial proximity. The walker propels, and the fluorescence recovers. The final fluorescence intensity of the reaction mixture is related to the concentration of the target virus. The detection limit of this proposed method is 6.6 × 104 copies/mL for EV71 and 4.3 × 104 copies/mL for CVB3, respectively. Besides, this method was applied in detection of EV71 in clinical samples with a satisfactory result. The entire experiment is easy to operate, and the proposed method has great potential for practical use.

Novel Method of Clickable Quantum Dot Construction for Bioorthogonal Labeling.

Bioorthogonal chemistry has been considered as a powerful tool for biomolecule labeling due to its site specificity, moderate reaction conditions, high yield, and simple post-treatment. Covalent coupling is commonly used to modify quantum dots (QDs) with bioorthogonal functional group (azide or cycloalkyne), but it has a negative effect in the decrease of QDs' quantum yield and stability and increase of QDs' hydrodynamic diameter. To overcome these disadvantages, we propose a novel method for the preparation of two kinds of clickable QDs by the strong interaction of -SH with metal ions. One system involves azide-DNA-functionalized QDs, which are used for bioconjugation with dibenzocyclooctyne (DBCO)-modified glucose oxidase (GOx) to form a GOx-QDs complex. After bioconjugation, the stability of QDs was improved, and the activity of GOx was also enhanced. The GOx-QDs complex was used for rapid detection of blood glucose by spectroscopy, naked eye, and paper-based analytical devices. The second system involves DBCO-DNA-functionalized QDs, which are used for an in situ bioorthogonal labeling of HeLa cells through metabolic oligosaccharide engineering. Therefore, these clickable QDs based on DNA functionalization can be applied for rapid and effective labeling of biomolecules of interest.

Target-Induced Cascade Amplification for Homogeneous Virus Detection.

Detection of viruses with high sensitivity is critical for the prevention and treatment of the related disease. Two homogeneous target-induced cascade amplification methods were proposed for the detection of enterovirus 71 and coxsackievirus B3. These methods both employ DNAzyme but differ in the way in which the DNAzyme is amplified. In the hybridization chain reaction (HCR)-based strategy, the DNAzyme is assembled by hairpin DNA strands, while in the rolling circle amplification (RCA)-based strategy, the DNAzyme is synthesized by the polymerase. On the basis of the virion structure, we investigated the effects of using only VP1-antibody or VP1-antibody and VP2-antibody on the detection. And the combination of two kinds of antibodies was found to further improve the performance of the detection. Subsequently, the simultaneous detection of EV71 and CVB3 was achieved by the RCA-based strategy. And the proposed methods were also applied in clinical samples analysis with a satisfactory result, showing great potential for applications in virus detection.

Self-assembled fluorescent Ce(â ¢) coordination polymer as ratiometric probe for HIV antigen detection.

The viral capsid protein p24 of human immunodeficiency virus is expressed at different level during viral invasion. Detection of p24 is of great importance in acquired immunodeficiency syndrome monitoring and therapy. A ratiometric probe that is easily-synthesized was constructed based on self-assembled fluorescent Ce(Ⅲ) and fluorescein. Fluorescein was used as reference. Hydrogen peroxide quenches the fluorescence of the Ce(III) easily but does not quench the fluorescence of fluorescein. The mechanism of reaction was discussed. Benefiting from the sensitive response to hydrogen peroxide, this probe was applied for p24 detection in enzyme linked immunoassay. The fluorescence ratio was in a good linear relationship with the concentration of p24, and the detection limit was 1.1 pg mL-1. This proposed method has shown potential in virus detection with easy operation.

A fluorometric turn-on aptasensor for mucin 1 based on signal amplification via a hybridization chain reaction and the interaction between a luminescent ruthenium(II) complex and CdZnTeS quantum dots.

A fluorometric method is described for the determination of the tumor biomarker mucin 1 (MUC1). It is based on signal amplification of the hybridization chain reaction (HCR), and the interaction between a luminescent ruthenium(II) complex and CdZnTeS quantum dots (QDs). If MUC1 bind to the biotin-labeled aptamer, it will initiate HCR with hairpins H1 and H2 to form a long-range dsDNA. The long nucleic acid chains are then linked on the surface of streptavidin-modified magnetic microparticles (MMPs) through streptavidin-biotin interaction. The luminescent ruthenium(II) complex is then embedded in the long dsDNA linked to the MMPs. Hence, there is little Ru complex in the supernatant after magnetic separation, and the fluorescence of the CdZnTeS QDs (best measured at excitation/emission wavelengths of 350/530 nm) is only slightly quenched. In the absence of target, the fluorescence of the CdZnTeS QDs is strongly quenched. Fluorescence increases linearly in the 0.2-100 ng·mL-1 MUC1 concentration range, and the LOD is 0.13 ng·mL-1 (at S/N = 3). The method was applied to the determination of MUC1 in spiked human serum samples. Graphical abstract A fluorometric turn-on aptasensor for mucin 1 is described that is based on the interaction between a Ru(II) complex and quantum dots (QDs). The detection system includes biotin-labeled aptamer-H0, hairpins H1 and H2, streptavidin-modified magnetic microparticles (MMPs), Ru(bpy)2(dppx)2+ and CdZnTeS QDs.

In situ synthesis of photoluminescence-quenching nanopaper for rapid and robust detection of pathogens and proteins.

A green and facile method is presented for in situ synthesis of a novel photoluminescence-quenching nanopaper with a highly-efficient quenching ability, rapid reaction time and long-term storage. The as-prepared nanopaper is further used to construct an aptasensor platform with high performance, rapidness and robustness.

The behavior of a bipedal DNA walker moving on the surface of magnet microparticles and its application in DNA detection.

In this work, a three-dimensional DNA machine based on the isothermal strand-displacement polymerase reaction (ISDPR) has been constructed. The walking behavior of a DNA walker on the obstructive surface of magnetic beads has also been studied by adding different nucleic acid blocks. The "leg" of the DNA walker could hybridize with a hairpin structure DNA named H1 and lead to the opening of it. And the newly exposed stem would interact with a primer. A strand exchange has happened with the assistance of polymerase and dNTPs, so that the "leg" has been displaced and the DNA walker could be pushed to move on the surface. But the nucleic acid blocks could increase steric hindrance and obstruct this process, which is similar to the behavior of human beings walking on craggy paths. Through changing these blocks, such as the structure, the amount, and the length of blocks, the movement of the DNA walker has been controlled. What's more, the results of its application for DNA detection are satisfactory. The limit of detection is 21.6 pM. Also, this method has been successfully applied in complex biological samples. Graphical abstract ᅟ.

Rational construction of a DNA nanomachine for HIV nucleic acid ultrasensitive sensing.

HIV nucleic acids, one kind of significant biomarker, play an important role in fundamental studies and clinical diagnosis. Importantly, the early accurate diagnosis for HIV nucleic acids at ultralow concentrations can potentially extend the life of patients. In the current work, we developed a DNA nanomachine on gold nanoparticles (AuNPs) coupling rolling circle amplification and DNA walker cascade amplification for ultrasensitive detection of HIV nucleic acids. This DNA nanomachine sensing strategy exhibits a significantly low detection limit down to 1.46 fM. Furthermore, this DNA nanomachine biosensor is capable of detecting target DNA in real samples because of its high selectivity and sensitivity. Moreover, the DNA nanomachine biosensor is capable of discriminating single-base mismatch lower than 3.5 pM. The results showed that this DNA nanomachine biosensor has the potential for biomedical studies and clinical applications.

Facile Design of Phase Separation for Microfluidic Droplet-Based Liquid Phase Microextraction as a Front End to Electrothermal Vaporization-ICPMS for the Analysis of Trace Metals in Cells.

The issue of quantifying trace metals in cells has drawn widespread attention but is threatened with insufficient sensitivity of the instruments, complex cellular matrix and limited cell consumption. In this study, microfluidic droplet-based liquid phase microextraction (LPME), as a miniaturized platform, was developed and combined with electrothermal vaporization (ETV)-inductively coupled plasma mass spectrometry (ICPMS) for the analysis of trace Cd, Hg, Pb, and Bi in cells. A novel and facile design of phase separation region was proposed, which made the phase separation very easily for subsequent ETV-ICPMS detection. Mechanism of the phase separation was carefully discussed using the incompressible formulation of the Navier-Stokes equations. The developed microfluidic droplet-based LPME system exhibited much higher extraction efficiency to target metals than microfluidic stratified flow-based LPME. Under the optimized conditions, the limits of detection of the proposed microfluidic droplet-based LPME-ETV-ICPMS system were 2.5, 3.9, 5.5, and 3.4 ng L-1 for Cd, Hg, Pb, and Bi, respectively. The accuracy of the developed method was well validated by analyzing the target metals in Certified Reference Materials of GBW07601a human hair. Finally, the proposed method was successfully applied to the analysis of target metals in HeLa and HepG2 cells with the recoveries for the spiked samples ranging from 83.5 to 112.3%. Overall, the proposed design is a simple and reliable solution for the phase separation on droplet-chip and the microfluidic droplet-based LPME-ETV-ICPMS combination strategy shows great promise for trace elements analysis in cells.

Digital analysis with droplet-based microfluidic for the ultrasensitive detection of ß-gal and AFP.

In this study, we presented a digital analysis with droplet-based microfluidic, which was applied for the detection of ß-galactosidase (ß-Gal) and AFP. The ß-Gal was quantified according to the Poisson Distribution in digital analysis with droplets containing ß-Gal and fluorogenic substrate fluorescein di-ß-D-galactopyranoside (FDG). In our method, the lowest concentration of ß-Gal we could accurately detect was 5 fM. We found that the digital detection could be applied in quantifying protein biomarkers, take AFP for model sample. The AFP was detected through indirect detection with excess streptavidin-conjugated ß-galactosidase (SA-ß-gal) as signal tag. To be specific, firstly SA-ß-gal was conjugated to biotin-labeled AFP antibody, and then the unbounded signal tag in the reaction solution was taken out for digital detection with droplet-based microfluidic. As a result, the AFP could be also indirectly quantified at an fM level through subtraction. This experimental result demonstrated that the indirect detection with droplet-based digital analysis was able to detect biomarkers at a low level with a few samples on an ordinary flow-focusing microfluidic chip.

[Identification of isoparaffin components in petroleum middle fractions].

The identification of isoparaffin components in petroleum middle fractions including kerosene and diesel fuels was investigated by gas chromatography-mass spectrometry (GC/MS). The isodewaxing middle fraction and distilled diesel were selected as the objective samples for identification. It was shown that the isoparaffin components in middle fraction were well separated with their branched alkyl substituent numbers on a capillary chromatographic column in selected ion monitoring (SIM) mode of GC/MS. The identification for C10-C24 isoparaffins was realized with the technique of GC/MS, such as the fragmentation pathways of electron ionization and SIM technique, boiling point rule, published retention indices and theoretical rules about component retention behavior in GC including carbon number rule etc. Finally, the retention indices for the mono-substituted, di-substituted and multi-substituted isoparaffins from C10 to C24 were presented, which could provide an overall knowledge of isoparaffin distribution at carbon number level in fuels. Meanwhile, the peaks that could be well resolved in each isoparaffin group were also identified, and the detailed data for about 80 C10-C21 methyl-substituted isoparaffins and isoprenoid biomarkers were also given. The results showed that in isodewaxing middle fraction studied, the mono-substituted and di-substituted isoparaffins were the main paraffins, whereas in distilled diesel studies, the mono-substituted isoparaffins and isoprenoid biomarkers were the main ones.

[Recent development in petroleomics based on the high-resolution mass spectrometry].

As the increase of the remaining reserves of the heavier/sourer crude oil and the requirement of the clean energy, it is significant to sufficiently characterize the molecular composition of the petroleum for the selection of the refining processes and the realization of the economic value of the crude oil. Petroleomics, which is based on the high-resolution mass spectrometry platform, is a powerful tool to achieve this goal. In this paper, the analytical technology applied in petroleomics and its most recent development are reviewed, and the perspective of petroleomics is also discussed.

[Roles of type II 11ß-hydroxysteroid dehydrogenase and its signaling factors in pathogenesis of persistent pulmonary hypertension in neonatal rats].

OBJECTIVE: To study the roles of type II 11ß-hydroxysteroid dehydrogenase (11ß-HSD2) and it's signaling factors in the lung tissue in pathogenesis of persistent pulmonary hypertension (PPH) in neonatal rats. METHODS: Six Sprague-Dawley rats on the 19th day of pregnancy were randomly divided into PPH and control groups (n=3 each). The PPH group was intraperitoneally injected with indomethacin (0.5 mg/kg) twice daily and exposed in 12% oxygen for three days, in order to prepare a fetal rat model of PPH. The control group was intraperitoneally injected with an equal volume of normal saline and exposed to air. Neonatal rats were born by caesarean section from both groups on the 22nd day of pregnancy. In each group, 15 neonatal rats were randomly selected and sacrificed. 11ß-HSD2 expression in the lung tissue of neonatal rats were observed by Confocal laser technology, and serum cortisol levels and prostacyclin, renin, angiotensin and aldosterone in the lung tissue of both groups were measured using ELISA. RESULTS: 11ß-HSD2 protein was widely expressed in the lung tissue of the control and PPH groups. The levels of 11ß-HSD2 and prostacyclin in the lung tissue were lower in the PPH group than in the control group, while serum cortisol levels and renin, angiotensin and aldosterone in the lung tissue were higher in the PPH group than in the control group (P<0.05). CONCLUSIONS: 11ß-HSD2 and it's signaling factors play roles in pathogenesis of PPH in neonatal rats.

[[Molecular composition of saturated hydrocarbons in diesels by comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry]].

An analytical method for separation and identification of the saturated hydrocarbons in diesels at molecular level by comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC x GC-TOF MS)was established. The saturated hydrocarbons were pre-separated from diesel samples by solid phase extraction before GC x GC-TOF MS analysis. More than 1,000 individual compounds (including paraffins, naphthenes and ole- fins) in coker diesel were tentatively identified based on NIST library search, mass spectrum resolution, boiling point distribution law and separation characteristics. Normal paraffins showed great regularity and could be identified easily through the relative position with pristane and phytane. The cyclic alkanes arranged above paraffins with the increasing number of rings. The normal alkyl cyclohexanes and cyclopentanes were well distinguished due to the difference of their polarity. Normal α-olefins which were often neglected in the past were also identified. With the support of the above-introduced identification, the distribution by structural type and carbon number were presented using peak area normalization. This analytical method was suc- cessfully used to investigate the molecular composition of saturated fractions in different diesel samples. All the results indicated that the molecular compositions of saturates in catalytic cracking diesel and coker diesel were significantly different because of the processing mechanism. This method provided technical support for the characterization of saturated hydrocar- bons in diesels and the investigation of processing mechanism.

The identification of highly similar crude oils by infrared spectroscopy combined with pattern recognition method.

Based on attenuated total reflectance mid-infrared spectroscopy and moving window correlation coefficient method, a efficient analytical tool is presented for rapid identification of crude oil type. Spectra between highly similar crude oils can be easily distinguished by the spectral searching method. Compared with NIR, IR method can distinguish highly similar mixed crude oils given in the study. Parameters of the moving window correlation coefficient method are studied in the paper.

[Application of gas chromatography in research of biodiesel processing].

Applications of gas chromatography in the research of biodiesel processing are reviewed with 27 references, including the analysis of fatty acid methyl esters (FAME) in the reaction products and final biodiesel, the determination of mono-, di- and tri-glycerides, the contents and distribution of free fatty acids, and the determination of trace methanol in biodiesel. The effects of various factors for analysis of the reaction products are discussed, such as injection mode, column type and silylation. A method for the determination of trace methanol in biodiesel products with dual-columns and pressure backflush system is proposed. 1-Propanol was used as the internal standard. After methanol and 1-propanol entered the analytical column through pre-column, the pressure was changed to backflush the heavy components through the split vent. A polar PEG-20M column was applied for the analysis of the contents and distribution of FAME in biodiesels from 8 different vegetable oils.

[Determination of naphthenic acids in distillates of crude oil by gas chromatography/chemical ionization-mass spectrometry].

The petroleum carboxylic acids in 200-420 degrees C distillate of crude oil were separated by the extraction with column chromatography on an anion exchange resin. The effect of the composition and structure of naphthenic acids on separation were studied by the infra-red (IR) spectroscopic techniques. Naphthenic acids and iso-butane reagent gas were introduced into the ion source for chemical ionization, in which the ions represented by [M + C4H9]+ were used to calculate the relative molecular mass for each acid. Based on the mass spectra of pure fatty and naphthenic acids, in combination with the z-series formula CnH(2n + z)O2, the naphthenic acids can be classified into fatty, mono-, bi- ... hexa-cyclic types. The results indicated that the relative molecular mass range of naphthenic acids in this distillates was 170-510, and the carbon number range was C10-C35. The contents of bi-cyclic and tri-cyclic naphthenic acids were higher than others.