Vision rescue via unconstrained in vivo prime editing in degenerating neural retinas.

Retinitis pigmentosa (RP) is an inherited retinal dystrophy causing progressive and irreversible loss of retinal photoreceptors. Here, we developed a genome-editing tool characterized by the versatility of prime editors (PEs) and unconstrained PAM requirement of a SpCas9 variant (SpRY), referred to as PESpRY. The diseased retinas of Pde6b-associated RP mouse model were transduced via a dual AAV system packaging PESpRY for the in vivo genome editing through a non-NGG PAM (GTG). The progressing cell loss was reversed once the mutation was corrected, leading to substantial rescue of photoreceptors and production of functional PDE6ß. The treated mice exhibited significant responses in electroretinogram and displayed good performance in both passive and active avoidance tests. Moreover, they presented an apparent improvement in visual stimuli-driven optomotor responses and efficiently completed visually guided water-maze tasks. Together, our study provides convincing evidence for the prevention of vision loss caused by RP-associated gene mutations via unconstrained in vivo prime editing in the degenerating retinas.

Roles of Histone Acetyltransferases and Deacetylases in the Retinal Development and Diseases.

The critical role of epigenetic modification of histones in maintaining the normal function of the nervous system has attracted increasing attention. Among these modifications, the level of histone acetylation, modulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), is essential in regulating gene expression. In recent years, the research progress on the function of HDACs in retinal development and disease has advanced remarkably, while that regarding HATs remains to be investigated. Here, we overview the roles of HATs and HDACs in regulating the development of diverse retinal cells, including retinal progenitor cells, photoreceptor cells, bipolar cells, ganglion cells, and Müller glial cells. The effects of HATs and HDACs on the progression of various retinal diseases are also discussed with the highlight of the proof-of-concept research regarding the application of available HDAC inhibitors in treating retinal diseases.

Modification of Microfluidic Paper-Based Devices with an Oxidant Layer for Distance Readout of Reducing Substances.

We developed a novel strategy for modification of paper cellulose with water-insoluble oxidants for distance readout of reducing substances on microfluidic paper-based analytical devices (µPADs). Water-insoluble oxidants were formed and modified onto paper cellulose through the redox reaction that occurred between paper cellulose and potassium permanganate deposited on the paper channel, developing a yellowish-brown color on the channel. As aqueous solutions containing reducing substances flowed along the channel, reducing substances were consumed owing to the redox reaction that occurred between oxidants and reducing substances until the reducing substances were depleted, forming a discolored zone on the yellowish-brown channel. The redox reaction between insoluble oxidants and reducing substances on the paper cellulose could be used for distance-based detection of a wide variety of reducing substances, which is similar to the classical potassium permanganate titration that employs the redox reaction that occurred between potassium permanganate and reducing substances. We believe that this method will broaden the analytical applications of distance-based detection on µPADs. This method was applied to ascorbic acid assay and captopril assay in real samples with analytical results comparing well with the labeled values, demonstrating its great potential in real sample analysis.

Sample injection and electrophoretic separation on a simple laminated paper based analytical device.

We described a strategy to perform multistep operations on a simple laminated paper-based separation device by using electrokinetic flow to manipulate the fluids. A laminated crossed-channel paper-based separation device was fabricated by cutting a filter paper sheet followed by lamination. Multiple function units including sample loading, sample injection, and electrophoretic separation were integrated on a single paper based analytical device for the first time, by applying potential at different reservoirs for sample, sample waste, buffer, and buffer waste. As a proof-of-concept demonstration, mixed sample solution containing carmine and sunset yellow were loaded in the sampling channel, and then injected into separation channel followed by electrophoretic separation, by adjusting the potentials applied at the four terminals of sampling and separation channel. The effects of buffer pH, buffer concentration, channel width, and separation time on resolution of electrophoretic separation were studied. This strategy may be used to perform multistep operations such as reagent dilution, sample injection, mixing, reaction, and separation on a single microfluidic paper based analytical device, which is very attractive for building micro total analysis systems on microfluidic paper based analytical devices.

Defining microchannels and valves on a hydrophobic paper by low-cost inkjet printing of aqueous or weak organic solutions.

We describe a simple and cost-effective strategy for rapid fabrication of microfluidic paper-based analytical devices and valves by inkjet printing. NaOH aqueous solution was printed onto a hydrophobic filter paper, which was previously obtained by soaking in a trimethoxyoctadecylsilane-heptane solution, allowing selective wet etching of hydrophobic cellulose to create hydrophilic-hydrophobic contrast with a relatively good resolution. Hexadecyltrimethylammonium bromide (CTMAB)-ethanol solution was printed onto hydrophobic paper to fabricate temperature-controlled valves. At low temperature, CTMAB deposited on the paper is insoluble in aqueous fluid, thus the paper remains hydrophobic. At high temperature, CTMAB becomes soluble so the CTMAB-deposited channel becomes hydrophilic, allowing the wicking of aqueous solution through the valve. We believe that this strategy will be very attractive for the development of simple micro analytical devices for point-of-care applications, including diagnostic testing, food safety control, and environmental monitoring.

Fabrication of a microfluidic paper-based analytical device by silanization of filter cellulose using a paper mask for glucose assay.

We developed a novel, low-cost and simple method for the fabrication of microfluidic paper-based analytical devices (µPADs) by silanization of filter cellulose using a paper mask having a specific pattern. The paper mask was penetrated with trimethoxyoctadecylsilane (TMOS) by immersing into TMOS-heptane solution. By heating the filter paper sandwiched between the paper mask and glass slides, TMOS was immobilized onto the filter cellulose via the reaction between cellulose OH and TMOS, while the hydrophilic area was not silanized because it was not in contact with the paper mask penetrated with TMOS. The effects of some factors including TMOS concentration, heating temperature and time on the fabrication of µPADs were studied. This method is free of any expensive equipment and metal masks, and could be performed by untrained personnel. These features are very attractive for the fabrication and applications of µPADs in developing countries or resource-limited settings. A flower-shaped µPAD was fabricated and used to determine glucose in human serum samples. The contents determined by this method agreed well with those determined by a standard method.

Analysis of intracellular reducing levels in human hepatocytes on three-dimensional focusing microchip.

A novel three-dimensional hydrodynamic focusing microfluidic device integrated with high-throughput cell sampling and detection of intracellular contents is presented. It has a pivotal role in maintaining the reducing environment in cells. Intracellular reducing species such as vitamin C and glutathione in normal and tumor cells were labeled by a newly synthesized 2,2,6,6-tetramethyl-piperidine-1-oxyl-based fluorescent probe. Hepatocytes are adherent cells, which are prone to attaching to the channel surface. To avoid the attachment of cells on the channel surface, a single channel microchip with three sheath-flow channels located on both sides of and below the sampling channel was developed. Hydrostatic pressure generated by emptying the sample waste reservoir was used as driving force of fluid on the microchip. Owing to the difference between the liquid levels of the reservoirs, the labeled cells were three-dimensional hydrodynamically focused and transported from the sample reservoir to the sample waste reservoir. Hydrostatic pressure takes advantage of its ease of generation on a microfluidic chip without any external pressure pump, which drives three sheath-flow streams to constrain a sample flow stream into a narrow stream to avoid blockage of the sampling channel by adhered cells. The intracellular reducing levels of HepG2 cells and L02 cells were detected by home-built laser-induced fluorescence detector. The analysis throughput achieved in this microfluidic system was about 59-68 cells/min.

Determination of chlorobenzenes in water samples by solid-phase disk extraction and gas chromatography-electron capture detection.

A simple, rapid, sensitive and high throughput method is described, based on solid-phase disk extraction (SPDE) and gas chromatography-electron capture detection, for the determination of chlorobenzens (CBs) in water samples. The proposed SPDE sample pretreatment method was initially optimized and the optimum experimental conditions were found to be as follows: 500 mL water sample (pH 2.5) extracted and enriched by an Empore 3-stn C18 (octadecyl) SPE disk at flow rate of 5 to 50 mL/min, eluted by 5 mL of acetone and 3 × 5 mL of methylene dichloride. The linearity of the method ranged from 0.02 to 0.4 µg/L for dichlorobenzene isomers, 0.0022-0.044 µg/L for trichlorobenzene isomers, 0.005-0.01 µg/L for tetrachlorobenzene isomers and 0.00025 to 0.005 µg/L for pentachlorobenzenes and hexachlorobenzenes, with correlation coefficients ranging between 0.9991 and 0.9999. The limits of detection were in the low ng/L level, ranging between 0.05 and 4 ng/L. The recoveries of spiked CBs with the external calibration method at different concentration levels in deionized/distilled water, tap water and sea water samples were 99-115, 91-106% and 96-110%, respectively, and with relative standard deviations of 4.5-7.6, 4.2-6.8 and 3.6-6.6% (n = 5), respectively. It is concluded that this method can successfully be applied for the determination of CBs in deionized/distilled water, tap water and sea water samples.

A simple paper-based sensor fabricated by selective wet etching of silanized filter paper using a paper mask.

We developed a novel strategy for fabrication of microfluidic paper-based analytical devices (µPADs) by selective wet etching of hydrophobic filter paper using a paper mask having a specific design. The fabrication process consists of two steps. First, the hydrophilic filter paper was patterned hydrophobic by using trimethoxyoctadecylsilane (TMOS) solution as the patterning agent. Next, a paper mask penetrated with NaOH solution (containing 30% glycerol) was aligned onto the hydrophobic filter paper, allowing the etching of the silanized filter paper by the etching reagent. The masked region turned highly hydrophilic whereas the unmasked region remains highly hydrophobic. Thus, hydrophilic channels, reservoirs, and detection zones were generated and delimited by the hydrophobic barriers. The effects of some factors including TMOS concentration, etching temperature, etching time, and NaOH concentration on fabrication of µPAD were studied. Being free of any expensive equipment, metal mask and expensive reagents, this rapid, simple, and cost-effective method could be used to fabricate µPAD by untrained personnel with minimum cost. A flower-shaped µPAD fabricated by this presented method was applied to the glucose assay in artificial urine samples with good performance, indicating its feasibility as a quantitative analysis device. We believe that this method would be very attractive to the development of simple microfluidic devices for point-of-care applications in clinical diagnostics, food safety, and environmental protection.

Rapid determination of catecholamines in urine samples by nonaqueous microchip electrophoresis with LIF detection.

A method was developed for the rapid separation of catecholamines by nonaqueous microchip electrophoresis (NAMCE) with LIF detection, A homemade pump-free negative pressure sampling device was used for rapid bias-free sampling in NAMCE, the injection time was 0.5 s and the electrophoresis separation conditions were optimized. Under the optimized conditions, the samples were separated completely in <1 min. The average migration times of the epinephrine (E), dopamine (DA), and norepinephrine (NE) were 34.26, 43.81, and 50.07 s, with an RSD of 1.05, 1.26, and 0.89% (n = 7), respectively. The linearity of the method ranged from 0.0125 to 2.0 mg/L for E and 0.025~4.0 mg/L for DA and NE, with correlation coefficients ranging between 0.9978 and 0.9986. The detection limits of E, DA, and NE were 2.5, 5.0, and 5.0 µg/L, respectively. The recoveries of E, DA, and NE in spiked urine samples were between 86 and 103%, with RSDs of 4.5~6.8% (n = 5). The proposed NAMCE with LIF detection combined with a pump-free negative pressure sampling device is a simple, inexpensive, energy efficient, miniaturized system that can be successfully applied for the determination of catecholamines in urine samples.

Rapid determination of catecholamines in urine samples by nonaqueous microchip electrophoresis with LIF detection.

A method was developed for the rapid separation of catecholamines by nonaqueous microchip electrophoresis with LIF detection, A homemade pump-free negative pressure sampling device was used for rapid bias-free sampling in nonaqueous microchip electrophoresis, the injection time was 0.5 s and the electrophoresis separation conditions were optimized. Under the optimized conditions, the samples were separated completely in less than 1 min. The average migration times of the epinephrine, dopamine, and norepinephrine were 34.26, 43.81, and 50.07 s, with a relative standard deviation of 1.05, 1.26 and 0.89% (n = 7), respectively. The linearity of the method ranged from 0.0125 to 2.0 mg/L for epinephrine and 0.025∼4.0 mg/L for dopamine, and norepinephrine, with correlation coefficients ranging between 0.9978 and 0.9986. The detection limits of epinephrine, dopamine, and norepinephrine were 2.5, 5.0 and 5.0 µg/L, respectively. The recoveries of epinephrine, dopamine, and norepinephrine in spiked urine samples were between 86 and 103%, with relative standard deviations of 4.5∼6.8% (n = 5). The proposed nonaqueous microchip electrophoresis with laser induced fluorescence detection system combined with a pump-free negative pressure sampling device was a simple, inexpensive, energy efficient, miniaturized system that can be successfully applied for the determination of catecholamines in urine samples. This article is protected by copyright. All rights reserved.

(3R,4S,5R)-Methyl 3,5-bis-[(tert-butyl-dimethyl-sil-yl)-oxy]-4-meth-oxy-cyclo-hex-1-ene-carboxyl-ate.

The title compound, C21H42O5Si2, was synthesized from (3R,4S,5R)-methyl 3,5-bis-[(tert-butyl-dimethyl-sil-yl)-oxy]-4-hy-droxy-cyclo-hex-1-ene-carboxyl-ate by an esterification reaction. The cyclo-hexene ring adopts a half-chair conformation. In the crystal, mol-ecules are linked via C-H⋯O hydrogen bonds, forming helical chains propagating along [010].

Shape deformation and recovery of multilayer microcapsules after being squeezed through a microchannel.

The deformation and recovery behaviors of multilayer microcapsules were investigated after being forced to flow through a microchannel. The microchannel device with a constriction (5.7 µm in depth) in the middle was designed, and the multilayer microcapsules with different size and layer thickness (and thereby different mechanical strength) were used. Deformation in the microchannel was observed for all the capsules with a size larger than the constriction height, and its extent was mainly governed by the difference between capsule size and constriction height. The squeezed microcapsules could recover their original spherical shape when the deformation extent was smaller than 16%, whereas permanent physical deformation took place when the deformation extent was larger than 34%. The capsules filled with polyelectrolytes could greatly enhance their shape recovery ability due to the higher osmotic pressure in the capsule interior and could well maintain the preloaded low-molecular-weight dyes regardless of the squeezing.

Three-dimensional (3D) hydrodynamic focusing for continuous sampling and analysis of adherent cells.

A simple three-dimensional (3D) hydrodynamic focusing microfluidic device integrated with continuous sampling, rapid dynamic lysis, capillary electrophoretic (CE) separation and detection of intracellular content is presented. One of the major difficulties in microfluidic cell analysis for adherent cells is that the cells are prone to attaching to the channel surface. To solve this problem, a cross microfluidic chip with three sheath-flow channels located on both sides of and below the sampling channel was developed. With the three sheath flows around the sample solution-containing cells, the formed soft fluid wall prevents the cells from adhering to the channel surface. Labeled cells were 3D hydrodynamically focused by the sheath-flow streams and smoothly introduced into the cross-section one by one. The introduction of sheath-flow streams not only ensured single-cell sampling but avoided blockage of the sampling channel by adherent cells as well. The maximum rate for introduction of individual cells into the separation channel was about 151 cells min(-1). With electric field applied on the separation channel, the aligned cells were driven into the separation channel and rapidly lysed within 400 ms at the entry of the channel by sodium dodecylsulfate (SDS) added in the sheath-flow solution. The microfluidic system was evaluated by analysis of reduced glutathione (GSH) and reactive oxygen species (ROS) in single HepG2 cells. The average analysis throughput of ROS and GSH in single cells was 16-18 cells min(-1).

Continuous cell introduction and rapid dynamic lysis for high-throughput single-cell analysis on microfludic chips with hydrodynamic focusing.

A chip-based microfluidic system for high-throughput single-cell analysis is described. The system was integrated with continuous introduction of individual cells, rapid dynamic lysis, capillary electrophoretic (CE) separation and laser induced fluorescence (LIF) detection. A cross microfluidic chip with one sheath-flow channel located on each side of the sampling channel was designed. The labeled cells were hydrodynamically focused by sheath-flow streams and sequentially introduced into the cross section of the microchip under hydrostatic pressure generated by adjusting liquid levels in the reservoirs. Combined with the electric field applied on the separation channel, the aligned cells were driven into the separation channel and rapidly lysed within 33ms at the entry of the separation channel by Triton X-100 added in the sheath-flow solution. The maximum rate for introducing individual cells into the separation channel was about 150cells/min. The introduction of sheath-flow streams also significantly reduced the concentration of phosphate-buffered saline (PBS) injected into the separation channel along with single cells, thus reducing Joule heating during electrophoretic separation. The performance of this microfluidic system was evaluated by analysis of reduced glutathione (GSH) and reactive oxygen species (ROS) in single erythrocytes. A throughput of 38cells/min was obtained. The proposed method is simple and robust for high-throughput single-cell analysis, allowing for analysis of cell population with considerable size to generate results with statistical significance.

Simultaneous determination of trace iron and aluminum by catalytic spectrophotometry based on a novel oxidation reaction of xylene cyanol FF.

A new, simple, sensitive and selective method for the simultaneous determination of trace iron and aluminum by catalytic spectrophotometry was presented, based on the catalytic effects of iron and aluminum on the discoloring reaction of xylene cyanol FF proceeded by hydrogen peroxide and potassium periodate in weak nitric acid medium. No catalytic effect was obtained in the presence of hydrogen peroxide or potassium periodate only. With the conditional rate constants determined in reaction systems catalyzed by Al or Fe only, the concentrations of Fe and Al in the samples can be calculated. The method was applied to the simultaneous determination of trace Fe and Al in tap water, lake water, river water and tea leaves without separation and preconcentration.