Separation Resolution Research Articles

Improving the separation ability of MOF-based stationary phases by increasing the thermodynamic differentiation of analytes

For chromatographic separation, the reasonable modulation of stationary phases is the key factor to achieve high separation performance. We proposed that developing MOF stationary phases through precisely modulating the thermodynamic interactions between MOFs and analytes is conducive to improving the separation resolution. MIL-125, MIL-125-NH2, MIL-143-BTB, and MIL-143-TATB were developed as stationary phases with the careful modulation of organic ligands. MIL-125-NH2 and MIL-143-TATB coated columns exhibited much better separation performance than their counterparts, MIL-125 and MIL-143-BTB, respectively. The investigation of the separation mechanism indicated that thermodynamic interaction, rather than kinetic diffusion, was responsible for the separation improvement. MIL-125-NH2 and MIL-143-TATB provided stronger and distinguishable interactions with targets than corresponding MIL-125 and MIL-143-BTB, respectively, resulting in enhanced separation performance. This work demonstrates a guide to improving the separation performance of MOF stationary phases by increasing the thermodynamic interactions between MOFs and analytes.

In situ growth of L012-reduced gold nanoparticles-loaded graphitic carbon nitride nanocomposite for potential-resolved ratiometric electrochemiluminescence analysis

BackgroundMost electrochemiluminescence (ECL) analytical methods involve only changes in one single signal, which greatly limits their high sensitivity and stability for applied research. Potential-resolved electrochemiluminescence (PRECL), which provides calibrated analysis by self-correcting signal responses from dual channels, has attracted great interest in recent years. However, research on PRECL nanomaterials is still at an early stage. It is critical to develop a novel PRECL nanocomposite with high intensity in light emission, high resolution in potential separation, and reduced complexity in reaction systems for accurate detection. ResultsHere, a novel PRECL nanocomposite, L012-Au/g-C3N4, was synthesized by in situ growth of L012-reduced gold nanoparticles (L012-Au) on the surface of graphitic carbon nitride (g-C3N4) nanosheets. The g-C3N4 nanosheets served as effective carriers for L012 molecules and L012-Au, as well as efficient cathodic ECL emitters. With the addition of a single co-reactant (K2S2O8), two fully separated ECL peaks were observed around −1.3 V (ECL-1) and +0.6 V (ECL-2) at neutral conditions. The catalytic character of the incorporated gold nanoparticles accelerated the anodic ECL reaction and greatly enhanced the ECL intensity. Furthermore, an ECL mechanism based on the competitive relationship between K2S2O8 and dissolved oxygen was proposed. When dopamine (DA) was introduced into the PRECL reaction system, ECL-1 remained essentially unchanged, while ECL-2 intensity was significantly reduced. By analyzing the intensity ratio of ECL-2 to ECL-1 (I2/I1) to the detecting target, a ratiometric DA sensor with high sensitivity and stability was successfully constructed. SignificanceA nanocomposite L012-Au/g-C3N4 that can induce high-intensity ECL with two separated potentials in each polarity has been prepared by a simple synthetic method. The developed ratiometric sensor can eliminate systematic errors and improve accuracy. In addition, the multifunctionality of the nanocomposite and the necessity of only one co-reactant for neutral-condition ECL greatly reduced the reaction complexity. Our work offers new PRECL solutions to design highly sensitive and stable multi-response detection systems for in vitro diagnostics.

Construction of Asymmetric Filler Density Mixed-Matrix MOF Membranes via Liquid-Phase Ion Activation for Advanced Oxidation Processes.

Mixed-matrix membrane (MMM) reactors incorporating metal-organic framework (MOF) fillers have significant applications in fields such as separation, catalysis, and chiral resolution. However, the traditional method of directly mixing MOF fillers with polymers results in a symmetric structure where most of the MOF fillers are uniformly distributed across the membrane reactor's cross-section. During usage, this leads to a pronounced trade-off between flux and efficiency and the waste of the MOF, as the rich pore characteristics and active sites of MOFs are not fully utilized. In this study, we propose a liquid-phase ion activation strategy that enables the in situ formation of a uniform and densely packed MOF shell layer on the membrane surface during the phase inversion process of MMM fabrication. This asymmetric density distribution of the MOF-based MMM reactor selectively induces the generation of considerable nonradical 1O2 in the peroxomonosulfate (PMS) system, demonstrating superior catalytic degradation performance against antibiotics like TC and dye molecules (with a flux of 1880 L m-2 h-1 and a Kapp value of 648 min-1), as well as outstanding cycling stability and environmental tolerance. This discovery sheds light on the design and engineering of composite membrane materials and applications for advanced oxidation processes in water purification.

Rapid separation of bile acid isomers via ion mobility mass spectrometry by complexing with spiramycin.

Bile acid (BA) is one of the main active components of bile and has multiple isomers, the structure or content of its isomers often changes due to diseases and other health problems; thus, the accurate detection of BA isomers is very important. In this study, two groups of BA isomers of glycine-conjugated BAs and taurine-conjugated BAs were simultaneously separated and quantitatively analyzed by ion mobility mass spectrometry (IM-MS). Especially, baseline mobility separation between the isomers was achieved by the formation of binary complexes via simple interaction with spiramycin (SPM), for which a separation resolution (Rp-p) of 1.96was reached. Moreover, BA isomers were quantitatively analyzed, and the limit of detection (LOD) of absolute quantification for TCDCA/TUDCA and GUDCA/GCDCA/GHDCA was 0.514 and 0.611ng∙mL-1, respectively; the LODs for molar ratio ranges of relative quantification for TCDCA/TUDCA, GUDCA/GHDCA, and GCDCA/GHDCA were 1:18-30:1, 1:18-21:1, and 1:19-21:1, respectively. Additionally, BA isomers analyzed in pig bile powder and bear bile powder were measured, which were in good consistency with those labeled, revealing the differences in BA composition and content between the two powders. Finally, BA detection and recovery analyses were performed on serum samples, with a recovery rate of ≥73.69%, RSD of ≤6.8%, and SR (standard deviation of recoveries, the degree of difference between measured values and average recovery) of ≤1.27. Due to the simple, rapid, and lack of need for complex sample preparation and chromatographic separation, the proposed method can be an effective method for BA detection in practical samples.

Angstrom-Level Tuning in Confined Space of Metal-Organic Framework for Ultra-High Resolution in Gas Chromatographic Separation.

Optimizing the balance between thermodynamic interaction and kinetic diffusion is pivotal to obtaining high-performance gas chromatographic stationary phases. Here, three aluminum-based metal-organic frameworks featuring fym topology were chosen to achieve such balance by refined controlling the thermodynamic interactions toward analytes at angstrom level in a confined space. The CAU-10-H with the middle-sized channels (5.4 Å) provided weak interactions with xylenes because of the benzene ring around the channel, leading to the fastest diffusion. While the MIL-160 provided stronger interactions toward the analytes due to the abundance of O-heterocyclic sites of 2,5-furandicarboxylic acid ligands, resulting in slightly higher diffusion barriers. Thereby, although MIL-160 had a larger channel (5.9 Å) than CAU-10-H, the xylenes still diffused more slowly in MIL-160 than in CAU-10-H. The CAU-10-NH2 with the channel of 4.7 Å provided overstrong thermodynamic interactions and significant stereospecific blockade to the analytes because of the NH2 sites in the confined channels. These factors collectively contributed to achieving the lowest diffusion kinetics. The confined interactions were also proved by molecular dynamics simulation. Furthermore, the application indicated that MIL-160 exhibited the highest separation ability as a GC stationary phase among all reported materials. This strategy offers an approach for developing high-performance MOF stationary phases.

Investigation of induced electroosmotic flow in small-scale capillary electrophoresis devices: Strategies for control and reversal.

Electroosmotic flow (EOF) is the bulk flow of solution in a capillary or microchannel induced by an applied electric potential. For capillary and microchip electrophoresis, the EOF enables analysis of both cations and anions in one separation and can be varied to modify separation speed and resolution. The EOF arises from an electrical double layer at the capillary wall and is normally controlled through the pH and ionic strength of the background buffer or with the use of additives. Understanding and controlling the electrical double layer is therefore critical for maintaining acceptable repeatability during method development. Surprisingly, in fused silica capillaries at low pH, studies observe an EOF even though the capillary surface should be neutralized. Previous work has suggested the presence of an "induced electroosmotic flow" from radial electric fields generated across the capillary wall due to the separation voltage and grounded components external to the capillary. Using thin-wall (15µm) fused silica separation capillaries to facilitate the study of radial fields, we show that the EOF mobility depends on both the separation voltage and the location of external grounds. This is consistent with the induced EOF model, in which radial electric fields embed positive charges at the capillary walls to create an electrical double layer. The magnitude of the effect is characterized and shown to have long-range influences that are difficult to completely null by moving grounded components away from the separation capillary. Instead, active EOF control using externally applied potentials or a passive approach using a negative separation voltage are discussed as two possible methods for controlling the induced EOF. Both methods can reverse the EOF and improve the resolution and peak efficiency in amino acid separations.

Spiral microchannels with concave cross-section for enhanced cancer cell inertial separation.

Inertial microfluidic technologies have proven effective for particle focusing and separation in many microchannels, typically the channels with the rectangular and trapezoidal shapes. To advance particle focusing in complex channels, we propose a spiral channel combining rectangular and concave cross-sections for high-resolution particle and cell focusing and separation. Numerical simulations were conducted to illustrate the effects of channel geometry on secondary flow distribution and particle focusing positions. The simulation shows the concave cross-section generates two asymmetrical Dean vortices skewing towards the inner and outer channel walls, resulting to stronger flow velocity magnitudes near the walls than the channel center. Consequently, larger particles focus near the inner wall, while smaller particles are trapped closer to the outer wall under the influence of the stronger velocity magnitude near the walls. A microfluidic chip with the proposed channelgeometry, along with a traditional rectangular channel, was fabricated by 3D printing and PDMS casting. Fluorescent microbeads were used to investigate inertial focusing and separation behaviors in the microfluidicchips. Experimental results show that the concave channel facilitates particle focusing or trapping much closer to the walls than the traditional rectangular channel, achieving better separation resolution. Finally, the proposed channel was applied to separate lung cancer A549 cells from human blood, achieving a cancer cell recovery rate of ~ 84.78% (enrichment ratio over 820-fold) and a blood cell rejection rate of ~ 99.88%. This innovative channel design in inertial microfluidics offers new insights for enhanced particle focusing and holds significant promise for cell manipulation with improved separation resolution.

A numerical study of the effects of lateral flow and retention in open tubular vortex chromatography

Axial dispersion in chromatographic columns is responsible for a reduced separation efficiency. In the present research macrotransport theory is used to predict the phenomenological constants related to axial dispersion. We evaluate the efficacy of lateral flow induced by alternating current (AC) in the presence of retaining walls on the separation resolution. Results show that lateral flows induced by laterally applied potentials as low as 0.3 V reduce C-term dispersion by a factor of 5.0 for unretained conditions (k = 0) and 2.7 for retained (k = 5) conditions, with a diffusion coefficient (Dm) of 10–11m2/s. The present paper further contributes to the understanding of the use of secondary lateral flows for dispersion reduction and offers practical guidance for designing future vortex chromatographic columns. It appears that a maximal performance gain is attained at low aspect ratios (AR=1), with the gain reduced from a factor of 5 to 1.6 for AR=4 for unretained conditions, and from 2.7 to 1.4 for retained conditions (k = 5)

Separating the Living from the Dead: An Electrophoretic Approach.

Cell viability studies are essential in numerous applications, including drug development, clinical analysis, bioanalytical assessments, food safety, and environmental monitoring. Microfluidic electrokinetic (EK) devices have been proven to be effective platforms to discriminate microorganisms by their viability status. Two decades ago, live and dead Escherichia coli (E. coli) cells were trapped at distinct locations in an insulator-based EK (iEK) device with cylindrical insulating posts. At that time, the discrimination between live and dead cells was attributed to dielectrophoretic effects. This study presents the continuous separation between the live and dead E. coli cells, which was achieved primarily by combining linear and nonlinear electrophoretic effects in an iEK device. First, live and dead E. coli cells were characterized in terms of their electrophoretic migration, and then the properties of both live and dead E. coli cells were input into a mathematical model built using COMSOL Multiphysics software to identify appropriate voltages for performing an iEK separation in a T-cross iEK channel. Subsequently, live and dead cells were successfully separated experimentally in the form of an electropherogram, achieving a separation resolution of 1.87. This study demonstrated that linear and nonlinear electrophoresis phenomena are responsible for the discrimination between live and dead cells under DC electric fields in iEK devices. Continuous electrophoretic assessments, such as the one presented here, can be used to discriminate between distinct types of microorganisms including live and dead cell assessments.

Utilizing ESI-MS/MS and ion mobility spectrometry for structural characterization and isomer differentiation of 1, 2-unsaturated (1-4) linked disaccharide derivatives and their 2-C-functionalized analogues.

The 1, 2-unsaturated derivatives of (1-4) linked disaccharides serve as versatile building blocks for synthesizing biologically active compounds. Distinguishing between four pairs of stereoisomers in mixtures presents a challenging task. In this study, disaccharide derivatives are analyzed as alkali metal adducts using electrospray ionization tandem mass spectrometry (ESI-MS/MS), both as individual compounds and in mixtures by ion mobility mass spectrometry (IMS). Electrospray ionization (ESI) in combination with tandem mass spectrometry (MS/MS) in positive mode was employed to differentiate lithium adduct ions of hexa-acetyl/hexa-benzyl-D-lactals, and hexa-acetyl/hexa-benzyl-D-maltals, along with their corresponding 2-C-branched malonates. The high resolving power of trapped ion mobility spectrometry (TIMS) with the imeX™ functionality rapidly identified different metal adducts (Li, Na, Cs) as individual isomers and separated mixtures of stereoisomers. The measured collisional cross section (CCS) values were analyzed in relation to predicted CCS values. MS/MS spectra of the [M + Li]+ ions of glycal disaccharide analogues exhibited typical cross-ring and glycosidic bond cleavages. Collision-induced dissociation (CID) spectra provided insights into their fragmentation behavior, allowing differentiation of (1-4) linked disaccharides. TIMS technology delivered adjustable ion mobility resolution for suitable separation of the four sets of stereo isomeric compounds. However, accurately predicting CCS values to differentiate between respective isomeric pairs using the SigmaCCS program for sodium adducts is only partially achievable. ESI CID spectra of [M + Li]+ adduct ions for individual glycal disaccharide analogues facilitate the discrimination between alpha and beta (1-4) linked unsaturated disaccharides and their 2-C-branched analogues. Through optimized experimental conditions, complete baseline ion mobility separation of stereoisomer pairs of the Cs adducts is achieved.

Hydrophilic interaction liquid chromatography: An efficient tool for assessing thorium interaction with phosphorylated biomimetic peptides

In the field of nuclear toxicology, the knowledge of the interaction of actinides (An) with biomolecules is of prime concern in order to elucidate their toxicity mechanism and to further develop selective decorporating agents. In this work, we demonstrated the great potential of hydrophilic interaction liquid chromatography (HILIC) to separate polar thorium (Th) biomimetic peptide complexes, as a key starting point to tackle these challenges. Th4+ was used as plutonium (Pu4+) analogue and pS16 and pS1368 as synthetic di- and tetra-phosphorylated peptides capable of mimicking the interaction sites of these An in osteopontin (OPN), a hyperphosphorylated protein. The objective was to determine the relative affinity of pS16 and pS1368 towards Th4+, and to evaluate the pS1368 selectivity when Th4+ was in competition complexation reaction with UO22+ at physiological pH. To meet these aims, HILIC was simultaneously coupled to electrospray ionization mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS), which allowed to identify online the molecular structure of the separated complexes and quantify them, in a single step. Dedicated HILIC conditions were firstly set up to separate the new dimeric Th2(peptide)2 complexes with good separation resolution (peptide = pS16 or pS1368). By adding pS16 and pS1368 in different proportions relatively to Th4+, we found that lower or equal proportions of pS16 with respect to pS1368 were not sufficient to displace pS1368 from Th2pS13682 and pS16 proportion higher than pS1368 led to the formation of a predominant ternary complex Th2(pS16)(pS1368), demonstrating preferential Th4+ binding to the tetra-phosphorylated peptide. Finally, online identification and quantification of the formed complexes when Th4+ and UO22+ were mixed in equimolar ratio relatively to pS1368 showed that in spite of pS1368 has been specifically designed to coordinate UO22+, pS1368 is also Th4+-selective and exhibits stronger affinity for this latter than for UO22+. Hence, the results gathered through this approach highlight the impact of Th4+ coordination chemistry on its interaction with pS1368 and more widely to its affinity for biomolecules.

Simplified Synthesis of Poly(ethyleneimine)-Modified Silica Particles and Their Application in Oligosaccharide Isolation Methods.

There are great challenges in the field of natural product isolation and purification and in the pharmacological study of oligosaccharide monomers. And these isolation and purification processes are still universal problems in the study of natural products (NPs), traditional Chinese medicine (TCM), omics, etc. The same polymer-modified materials designed for the special separation of oligosaccharides, named Sil-epoxy-PEI and Sil-chloropropyl-PEI, were synthesized via two different methods and characterized by scanning electron microscopy combined with energy spectrum analysis, Fourier transform infrared spectroscopy, thermogravimetric analysis, zeta potential as well as surface area analysis, etc. Several nucleotide/nucleoside molecules with different polarities and selectivities were successfully isolated in our laboratory using stainless-steel columns filled with the synthesized material. In addition, the separation of saccharide probes and oligosaccharides mixtures in water extracts of Morinda officinalis were compared in HILIC mode. The results showed that the resolution of separations for the representative analytes of the Sil-epoxy-PEI column was higher than for the Sil-chloropropyl-PEI column, and the developed stationary phase exhibited improved performance compared to hydrothermal carbon, amide columns and other HILIC materials previously reported.

A simple and efficient approach for preparing cationic coating with tunable electroosmotic flow for capillary zone electrophoresis-mass spectrometry-based top-down proteomics

BackgroundCapillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has become a valuable analytical technique in top-down proteomics (TDP). CZE-MS/MS-based TDP typically employs separation capillaries with neutral coatings (i.e., linear polyacrylamide, LPA). However, issues related to separation resolution and reproducibility remain with the LPA-coated capillaries due to the unavoidable non-specific protein adsorption onto the capillary wall. Cationic coatings can be critical alternatives to LPA coating for CZE-MS/MS-based TDP due to the electrostatic repulsion between the positively charged capillary inner wall and proteoform molecules in the acidic separation buffer. Unfortunately, there are only very few studies using cationic coating-based CZE-MS/MS for TDP studies. ResultsIn this work, we aimed to develop a simple and efficient approach for preparing separation capillaries with a cationic coating, i.e., poly (acrylamide-co-(3-acrylamidopropyl) trimethylammonium chloride [PAMAPTAC]) for CZE-MS/MS-based TDP. The PAMAPTAC coating-based CZE-MS produced significantly better separation resolution of proteoforms compared to the traditionally used LPA-coated approach. It achieved reproducible separation and measurement of a simple proteoform mixture and a complex proteome sample (i.e., a yeast cell lysate) regarding migration time, proteoform intensity, and the number of proteoform identifications. The PAMAPTAC coating-based CZE-MS enabled the detection of large proteoforms (≥30 kDa) from the yeast cell lysate reproducibly without any size-based prefractionation. Interestingly, the mobility of proteoforms using the PAMAPTAC coating can be predicted accurately using a simple semi-empirical model. SignificanceThe results render the PAMAPTAC coating as a valuable alternative to the LPA coating to advance CZE-MS-based TDP towards high-resolution separation and highly reproducible measurement of proteoforms in complex samples.

Cascading in-situ grown UiO-66 and alternative calix[n]arenes for high-resolution isomer separation

High-resolution separation of numerous isomers is highly desirable for their high-quality analysis, yet challenging, which puts forward high requirements on the precise design of stationary phase. Herein, flexible 4-tert-butylcalix[n]arenes (TBCx[n]s) with different cavities have been intelligently integrated with in-situ grown UiO-66 coatings, to investigate their separation efficiencies for alkane and substituted benzene isomers. The modifications of UiO-66 with TBCx[n]s have made up for the deficiency of UiO-66 on the separation of ethylbenzene and styrene. The effects of TBCx[n]s on the separation performance of UiO-66@TBCx[n]s have demonstrated distinct regularities, i.e., the effects of TBCx[6] are superior to those of TBCx[8], while the TBCx[4] is the least effective. The baseline separation of all analytes has been achieved by the UiO-66@TBCx[6] column with enhanced selectivity and improved separation resolution (Rs). For the six groups of isomers that are most difficult to separate, the corresponding selectivity and Rs are greater than 1.09 and 5.48, respectively, confirming the outstanding separation performance of the UiO-66@TBCx[6] column and the feasibility of integrating soft supramolecule and rigid MOF.

Racetrack FAIMS for High-Resolution and High-Sensitivity Characterization of Peptide Conformers.

A racetrack field asymmetric waveform ion mobility spectrometry (r-FAIMS) device, which consists of both cylindrical FAIMS (c-FAIMS) and planar FAIMS (p-FAIMS) sections with a 1 mm gap width, was developed and applied for high-resolution and high-sensitivity exploration of conformational diversity for peptides. The optimal operating conditions of r-FAIMS were systemically studied, and the performance of the fully optimized r-FAIMS was compared to a previously developed p-FAIMS in detail by using pure nitrogen as the FAIMS carrier gas. Relying on the ion focusing effect in the c-FAIMS section, the intensity of the FAIMS spectrum for doubly charged bradykinin ions acquired by using r-FAIMS is ∼8.5-fold higher than that acquired by using p-FAIMS under the same resolving power/resolution condition, implying about an order of magnitude better sensitivity of r-FAIMS. In addition, the peak separation resolution of r-FAIMS was ∼1.70-fold higher than p-FAIMS under a similar sensitivity condition for doubly charged bradykinin ions. Due to a reduced gap width of the newly designed r-FAIMS (1 mm) as compared to the previously developed p-FAIMS (1.88 mm), r-FAIMS can operate at a much higher separation field with a similar FAIMS dispersion voltage (DV) to gain significantly higher resolving power. For triply charged syntide 2 ions, the resolving power of r-FAIMS can easily exceed 120 at -3.5 kV DV by using pure nitrogen as the FAIMS carrier gas as compared to 44.2 resolving power obtained by using p-FAIMS at -4.0 kV DV. All of the experimental results have confirmed that r-FAIMS can perform structural characterization of biomolecules with both high resolution and high sensitivity.

Acoustofluidic Virus Isolation via Bessel Beam Excitation Separation Technology.

The isolation of viruses from complex biological samples is essential for creating sensitive bioassays that assess the efficacy and safety of viral therapeutics and vaccines, which have played a critical role during the COVID-19 pandemic. However, existing methods of viral isolation are time-consuming and labor-intensive due to the multiple processing steps required, resulting in low yields. Here, we introduce the rapid, efficient, and high-resolution acoustofluidic isolation of viruses from complex biological samples via Bessel beam excitation separation technology (BEST). BEST isolates viruses by utilizing the nondiffractive and self-healing properties of 2D, in-plane acoustic Bessel beams to continuously separate cell-free viruses from biofluids, with high throughput and high viral RNA yield. By tuning the acoustic parameters, the cutoff size of isolated viruses can be easily adjusted to perform dynamic, size-selective virus isolation while simultaneously trapping larger particles and separating smaller particles and contaminants from the sample, achieving high-precision isolation of the target virus. BEST was used to isolate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from human saliva samples and Moloney Murine Leukemia Virus from cell culture media, demonstrating its potential use in both practical diagnostic applications and fundamental virology research. With high separation resolution, high yield, and high purity, BEST is a powerful tool for rapidly and efficiently isolating viruses. It has the potential to play an important role in the development of next-generation viral diagnostics, therapeutics, and vaccines.

A robust method for quantifying 42 phenolic compounds by RP-HPLC/DAD: Columns performance and characterization of Brazilian Citrus peels

Reliable analytical methods are the basis for the elucidation of phenolic compounds in foods. This study aimed to optimize and validate a method for determining 42 phenolics using reverse-phase (RP) high-performance liquid chromatography (HPLC) coupled to diode-array-detector-DAD. The performance of two RP columns was evaluated. The 150x4.6 mm 3-μm column showed superior separation quality, whereas 35 of the 42 phenolics showed a separation resolution ≥1.5. The method's linearity, precision (coefficient variation< 3.09%), recovery (87.5-103.2%), specificity, limits of detection (0.04-0.25 mg/L), and quantification (0.06-0.25 mg/L) had acceptable ranges. Thirty phenolics were quantified in Citrus peels, mainly flavanones, flavanols, flavonols, and phenolic acids, highlighting the high values of hesperidin (535-35070 mg/kg) and naringin (26-36466 mg/kg). Lemon peels named 'Lisboa,' 'Thaiti,' 'Thaiti-2000', and 'Thaiti-2001' presented the main phenolics associated with antioxidant capacity. The presented method was robust for determining 42 phenolic compounds, offering a new approach for bioactive compound quantification in food matrices.

Streamlining the purification of a clinical-grade oncolytic virus for therapeutic applications

Oncolytic viruses (OV) have emerged as a promising approach to mitigate the challenges of treating solid cancers. However, the lack of established manufacturing and downstream processing (DSP) platforms combined with the high treatment doses needed for clinical use (108–1011 TCID50/dose), hamper the widespread success of this therapeutic concept. Here, we present an efficient and scalable GMP-compliant process for the purification of a fusogenic oncolytic virus (rVSV-NDV). Non-GMO CCX.E10 cells grown in suspension in chemically defined medium were used for high titer rVSV-NDV batch production (3.2 × 108TCID50/mL) in stirred tank bioreactors. All DSP unit operations (DNA digestion, clarification, chromatography, TFF, and sterilizing filtration) were optimized to identify the best purification approach. Among several strategies evaluated, two filters enabled high throughput and turbidity reduction, while preventing any loss of infectious particles during the clarification step. For the intermediate purification, anion-exchange chromatography (AEX) was used in combination with the addition of polyprotic salts, which resulted in a maximum recovery yield of 86 % of infectious particles. The addition of citrate to the chromatography setup increased the separation resolution of rVSV-NDV particles from less negatively charged impurities. Global recovery yield after four operation units was 64 % with 99 % and 97 % of protein and DNA clearance, respectively. Together, with our previously optimized upstream process, we open up an avenue for large-scale manufacturing of oncolytic VSV-NDV for future clinical use.

Fluorescence lifetime imaging with distance and ranging using a miniaturised SPAD system

In this work we demonstrate a miniaturised imaging system based around a time-gated SPAD array operating in a “chip-on-tip” manner. Two versions of the system are demonstrated, each measuring 23 mm × 23 mm × 28 mm with differing fields of view and working distances. Initial tests demonstrate contrast between materials in widefield fluorescence imaging (WFLIm) mode, with frame rates of > 2 Hz achievable. Following this, WFLIm images of autofluorescence in ovine lung tissue are obtained at frame rates of ~ 1 Hz. Finally, the ability of the second system to perform simultaneous WFLIm and time of flight (aka Flourescence Lifetime Imaging Distance and Ranging, FLImDAR) is also tested. This shows that the system is capable of 4 mm resolution of object separation when tested on 3D printed samples. It is further demonstrated as being able to perform scene reconstruction on autofluorescent lung tissue. This system is, to date, the smallest chip on tip WFLIm system published, and is the first demonstration of the FLImDAR technique in a compact, portable system.

Preparation of carboxyl-functionalized silica core-shell microspheres and their applications in weak cation exchange chromatography, heavy metal removal, and lysozyme enrichment.

Chromatography is a technique of separation based on adsorption and/or interaction of target molecules with stationary phases. Herein, we report the design and fabrication of BTDA@SiO2 core-shell microspheres as a new class of stationary phase and demonstrate its impressive performance for chromatographic separations. The silica microspheres of BTDA@SiO2 were synthesized by in situ method with 1,3,5-benzenetricarboxaldehyde and 3,5-diaminobenzoic to separate peptides and proteins on high-performance liquid chromatography. The BTDA@SiO2 core-shell structure has a high specific surface area and retention factor of 4.27 and 8.31 for anionic and cationic peptides, respectively. The separation factor and resolution were high as well. A typical chromatogram illustrated nearly baseline resolution of the two peptides in less than 3 min. The BTDA@SiO2 was also highly stable in the pH range of 1 to 14. Furthermore, the prepared BTDA@SiO2 core-shell material not only be used for chromatographic separation but also as heavy metal removal from water. Using a BTDA@SiO2, we also achieved a lysozyme enrichment with a maximum saturated adsorption capacity reaching 714mg/g. In summary, BTDA@SiO2 has great application prospects and significance in separation and purification systems.