Hetero- and Homointerlocked Metal-Organic Cages.

Interlocked molecular assemblies constitute a captivating ensemble of chemical topologies, comprising two or more separate components that exhibit remarkably intricate structures. The interlocked molecular assemblies are typically identical, and heterointerlocked systems that comprise structurally distinct assemblies remain unexplored. Here, we demonstrate that metal-templated synthesis can be exploited to afford not only a homointerlocked cage but also a heterointerlocked cage. Treatment of a carboxylated 2,9-dimethyl-1,10-phenanthroline (dmp) or Cu(I) bis-dmp linker with a Ni4-p-tert-butylsulfonylcalix[4]arene cluster affords noninterlocked octahedron and quadruply interlocked double cages consisting of two identical tetragonal pyramids, respectively. In contrast, when a mixture of dmp and Cu(I) bis-dmp linkers is used, a quadruply heterointerlocked cage is produced, consisting of a tetragonal pyramid and an octahedron. With photoredox-active [Cu(dmp)2]+ in the structures, both interlocked cages exhibit remarkable performance as photocatalysts for atom transfer radical addition (ATRA) reactions of trifluoromethanesulfonyl chloride with alkenes or oxo-azidations of vinyl arenes. These interlocked structures serve the dual purpose of stabilizing photocatalytically active components against deactivation and encapsulating substrates within the cavity, resulting in yields comparable to or even surpassing those of their molecular counterparts. This work thus provides a new strategy that combines metal templating and nontemplating approaches to design new types of interlocked assemblies with intriguing architectures and properties.

Carbon nanodots combined with loop-mediated isothermal amplification (LAMP) for detection of African swine fever virus (ASFV).

The spread of African swine fever virus (ASFV) caused huge economic costs, so early detection is particularly important. Here, we established a fluorescence biosensor based on carbon nanodots (CNDs) and loop-mediated isothermal amplification (LAMP) to ultra-sensitively detect ASFV. LAMP with high efficiency produced a large amount of pyro phosphoric acid and caused pH change in a short time. CNDs with strong light stability had a large fluorescence response at the emission wavelength of 585.5 nm to small pH change by the excitation wavelength of 550 nm. The biosensor realized "turn-off-on" mode for ASFV detection with the detection limit as low as 15.21 copies µL-1. In addition, the biosensor had high accuracy in the actual sample assay. Therefore, the biosensor achieved rapid, sensitive, low-cost, and simple detection for ASFV. Moreover, the biosensor broadened the detection pathway of LAMP as a tool with great development prospect.

Non-nucleic acid extraction and ultra-sensitive detection of African swine fever virus via CRISPR/Cas12a.

Early diagnosis of the African swine fever virus (ASFV) is the main preventive measure for ASFV. Here, we developed a fluorescent biosensor and lateral flow assay (LFA) strip based on direct PCR combined with CRISPR/Cas12a system for ASF. Direct PCR can simultaneously split samples and efficiently amplify without sacrificing sensitivity, which eliminated the steps of nucleic acid extraction. Furthermore, by the CRISPR/Cas12a, the biosensor addressed false positives caused by non-specific amplification and had high sensitivity with the actual limit of detection (LOD) of 7.6×10-4 ng·µL-1 (4 copies·µL-1). In addition, the strategy was built on the lateral flow assay (LFA) strip to achieve visual and portable detection for point-of-care testing. Moreover, the biosensor by a fluorometer and LFA strip showed a high accuracy to rival qPCR in actual sample detection. Therefore, the biosensor is an ultra-sensitive and specific tool that can replace traditional methods. KEY POINTS: • No nucleic acid extraction, direct PCR-simplified steps, and reduced time and cost • CRISPR/Cas12a solved the false positives caused by nonspecific amplification • The combination of the LFA strip and biosensor is more convenient for POC detection.

Ultra-sensitive and point-of-care detection of Capripoxvirus (CaPV) based on loop-mediated amplification (LAMP) and trans-cleavage activity of CRISPR/Cpf1.

Capripoxvirus (CaPV) is one of the common skin diseases infecting cattle and sheep which can cause serious economic losses. Establishing ultra-sensitive, rapid, and point-of-care detection of CaPV is particularly important for hindering its spread. Here, we use the principle that CRISPR/Cpf1 can specifically recognize the target DNA and activate its trans-cleavage activity to identify the CaPV product amplified by loop-mediated amplification (LAMP). Under the designed specific primers, a set of LAMP which can amplify CaPV specifically was established and optimized firstly. Then, the CRISPR/Cpf1 was introduced to identify LAMP products. LAMP can be completed at a constant temperature, thus avoiding the use of temperature-variable instruments, making it possible to detect viruses outside the laboratory. To further satisfy the point-of-care detection of CaPV, we introduced a portable fluorometer and CRISPR-based lateral flow test. Due to the introduction of CRISPR/Cpf1, the sensitivity of the method is greatly increased, which is of great significance for the early detection of viruses. Through CRISPR/Cpf1-mediated fluorescence detection, we can detect CaPV as low as 1.47 × 10-3 TCID50 in 50 min, 1000 times more sensitive than quantitative real-time PCR. Through CRISPR-based lateral flow test, we can visually detect CaPV as low as 1.47 × 10-2 TCID50. Besides, this strategy can be used for the primary samples obtained from the cell culture of CaPV after simple ultrasonic disruption, which eliminates the complicated nucleic acid extraction steps required by traditional methods.

Selective excitation of four-wave mixing by helicity in gated graphene.

Gapless Dirac fermions in monolayer graphene give rise to an abundance of peculiar physical properties, including exceptional broadband nonlinear optical responses. By tuning the chemical potential, stacking order, and photonic structures, the effective modulation of nonlinear optical phenomena in graphene has been demonstrated in recent years. Here, we demonstrate that optical helicity can be used as an extra tuning knob for four-wave mixing in gated graphene. Our results reveal the helicity selection rule for four-wave mixing in monolayer graphene, revealing nearly perfect circular polarization. Corresponding theoretical interpretations of the helicity selection rule that are also applicable to other nonlinear optical processes and materials are presented.

Analysis of vaccine-like lumpy skin disease virus from flies near the western border of China.

Lumpy skin disease (LSD) is a devastating viral disease that occurs in cattle. In China, it was first detected in the Xin-Jiang autonomous region, near the border with Kazakhstan, in August 2019. As there were no new occurrences of LSD in either country following the first detection, the initial introduction of the virus remains unknown. Arthropod vectors were considered as potential vectors. Consequently, to identify the arthropod vectors involved in transmitting LSD virus (LSDV), an insect surveillance campaign was launched at four different sites scattered along the border, and samples from 22 flying insect species were collected and subjected to PCR assays. Following the Agianniotaki LSDV vaccine and Sprygin's general LSDV assays, two kinds of non-biting flies, namely, Musca domestica L and Muscina stabulans, were positive for LSDV. However, all the other insects tested negative. Viral DNA was only detected in wash fluid, implying body surface contamination of the virus. The negative test results suggest that non-biting flies are the dominant insects involved in the observed local epidemic. Three genomic regions encoding RPO30, GPCR, and LW126 were successfully sequenced and subjected to phylogenetic analysis. The sequences shared high homology with LSDV/Russia/Saratov/2017, a recombinant vaccine-like strain formerly identified in Russia, and clustered with LSDV vaccine strains in phylogenetic trees of RPO30 and LW126. However, the GPCR gene was seen to be solely clustered with LSDV field strains, implying differences in host affinity between these closely related vaccine-like strains. Despite this, there is no direct evidence to support cross-border transmission of the vaccine-like LSDV. To our knowledge, this is the first report of vaccine-like LSDV DNA detection in non-biting flies in China.

Artificial Biomolecular Channels: Enantioselective Transmembrane Transport of Amino Acids Mediated by Homochiral Zirconium Metal-Organic Cages.

Natural transport channels (or carriers), such as aquaporins, are a distinct type of biomacromolecule capable of highly effective transmembrane transport of water or ions. Such behavior is routine for biology but has proved difficult to achieve in synthetic systems. Perhaps most significantly, the enantioselective transmembrane transport of biomolecules is an especially challenging problem both for chemists and for natural systems. Herein, a group of homochiral zirconium metal-organic cages with four triangular opening windows have been proposed as artificial biomolecular channels for enantioselective transmembrane transport of natural amino acids. These structurally well-defined coordination cages are assembled from six synthetically accessible BINOL-derived chiral ligands as spacers and four n-Bu3-Cp3Zr3 clusters as vertices, forming tetrahedral-shaped architectures that feature an intrinsically chiral cavity decorated with an array of specifically positioned binding sites mediated from phenol to phenyl ether to crown ether groups. Fascinatingly, the transformation of single-molecule chirality to global supramolecular chirality within the space-restricted chiral microenvironments accompanies unprecedented chiral amplification, leading to the enantiospecific recognition of amino acids. By virtue of the highly structural stability and excellent biocompatibility, the orientation-independent cages can be molecularly embedded into lipid membranes, biomimetically serving as single-molecular chiral channels for polar-residue amino acids, with the properties that cage-1 featuring hydroxyl groups preferentially transports the l-asparagine, whereas cage-2 attaching crown ether groups spontaneously favor transporting d-arginine. We therefore develop a new type of self-assembled system that can potentially mimic the functions of transmembrane proteins in nature, which is a realistic candidate for further biomedical applications.

Efficient C2H2/CO2 Separation in Ultramicroporous Metal-Organic Frameworks with Record C2H2 Storage Density.

Physical separation of C2H2 from CO2 on metal-organic frameworks (MOFs) has received substantial research interest due to the advantages of simplicity, security, and energy efficiency. However, that C2H2 and CO2 exhibit very close physical properties makes their separation exceptionally challenging. Previous work appeared to mostly focused on introducing open metal sites that aims to enhance the C2H2 affinity at desired sites, whereas the reticular manipulation of organic components has rarely been investigated. In this work, by reticulating preselected amino and hydroxy functionalities into isostructural ultramicroporous chiral MOFs-Ni2(l-asp)2(bpy) (MOF-NH2) and Ni2(l-mal)2(bpy) (MOF-OH)-we targeted efficient C2H2 uptake and C2H2/CO2 separation, which outperforms most benchmark materials. Explicitly, MOF-OH adsorbs substantial amount of C2H2 with record storage density of 0.81 g mL-1 at ambient conditions, which even exceeds the solid density of C2H2 at 189 K. In addition, MOF-OH gave IAST selectivity of 25 toward equimolar mixture of C2H2/CO2, which is nearly twice higher than that of MOF-NH2. Notably, the adsorption enthalpies for C2H2 at zero converge in both MOFs are remarkably low (17.5 kJ mol-1 for MOF-OH and 16.7 kJ mol-1 for MOF-NH2), which to our knowledge are the lowest among efficient rigid C2H2 sorbents. The efficiencies of both MOFs for the separation of C2H2/CO2 are validated by multicycle breakthrough experiments. DFT calculations provide molecular-level insight over the adsorption/separation mechanism. Moreover, MOF-OH can survive in boiling water for at least 1 week and can be easily scaled up to kilograms eco-friendly and economically, which is very crucial for potential industrial implementation.

Fine-Tuning of Chiral Microenvironments within Triple-Stranded Helicates for Enhanced Enantioselectivity.

Here we report the formation of an unexpected and unique family of chiral helicates. Crystal structures show that these triple-stranded ZnII2 L3 complexes are held together by subcomponent assembly of axially chiral diamine-functionalized 1,1'-biphenol ditopic with 2-formylpyridine and ZnII . Specifically, the molecular helicity of the complexes can be controlled by the absolute configurations of the bimetallic vertices, which has been shown to be homoconfiguration (ΔΔ) or mesomeric configuration (ΔΛ), depending critically on the bulky groups and length of the spacers. Fascinatingly, in this system we can engineer the space-restricted chiral microenvironments with varied polar and apolar moieties, which profoundly influence the binding affinities and chiral discrimination properties of the helicates, leading to highly enantio- and helix-sense-selective recognition for chiral amino alcohols (up to 9.35). This work reveals the transformation of single-molecule chirality to global supramolecular chirality within well-defined helicates and demonstrates that their chiral discrimination are highly dependent on the superior microenvironments.

Development of multiplex oligonucleotide microarray for simultaneous detection of six swine pathogens.

In order to establish a high-throughput identification technique that simultaneously detects six major pathogens including APP, HPS, PRRSV, Mhp, PCV-2 and CSFV, six pairs of primers and probes were designed based on the specific conservative sequences of the pathogens, a multiplex PCR system was developed, hybrid parameters were optimized, and evaluation of the technology was performed. The results showed that the present detection method had a sensitivity of 5.8 × 102copies/µL for APP, 7.8 × 103 copies/µL for HPS, 6.8 × 103 copies/µL for Mhp, 6.3 × 102 copies/µL for PCV-2, 4.8 × 103 copies/µL for PRRSV, and 5.5 × 102 copies/µL for CSFV, respectively; and it produced no cross reaction against the other nine pathogens like swine-origin pseudorabies virus, porcine parvovirus, Japanese B encephalitis virus, swine vesicular disease virus, vesicular stomatitis virus, foot-and-mouth disease virus, bluetongue virus, peste des petits ruminants virus and salmonella. Application of the multiplex oligonucleotide microarray established here to testing 285 clinical blood samples indicated a single infection rate of 18.2 % (52/285) and a mixed infection rate of 6.3 % (18/285) which were consistent with the results of the sequencing verification. This technique might serve as a rapid and high-throughput method of detection for epidemic investigation and clinical diagnosis of multiple pathogens.

A Prototype VP-PET Imaging System Based on Highly Pixelated CdZnTe Detectors.

We investigated a prototype virtual-pinhole positron emission tomography (PET) system for small-animal imaging applications. The PET detector modules were made up of 1.3 mm lutetium-yttrium oxyorthosilicate (LYSO) arrays, and the insert detectors consisted of 0.6 mm pixelated cadmium zinc telluride (CdZnTe). To validate the imaging experiment, we did a Monte Carlo simulation for the virtual-pinhole PET (VP-PET) system in the Geant4 Application for Emission Tomography (GATE). For a point source of 22Na with a 0.5 mm diameter, the filtered back-projection algorithm-reconstructed PET image showed a resolution of 0.7 mm full-width-at-half-maximum. The system sensitivity was 0.46 cps/kBq at the center of the field view of the PET system with a source activity of 0.925 MBq and an energy window of 350 to 650 keV. A rod source phantom and a Derenzo phantom with 18F were also simulated to investigate the PET imaging ability. GATE simulation indicated that sources with 0.5 mm diameter could be clearly detected using 0.6 mm pixelated CdZnTe detectors as insert devices in a VP-PET system.

Study on the Relationship between Seed Absorbed Dose and Seed Composition of 252Cf Neutron Source Irradiated Bean Seed.

This study aims to further identify the biological effects of neutron-irradiated plants and provides insights into the mutation breeding of such plants. In this study, the neutron irradiation device designed by our institute was used to analyze the relationship between the seed components in different legume crops and their neutron absorption dose rate, fission gamma absorption dose rate, and induced gamma absorption dose rate. The results show that the effect sizes of the components on the neutron absorbed dose rate are as follows: ash > fat > moisture > carbohydrate > protein. The effect sizes of the components on the absorbed dose rate of fission gamma are as follows: ash > moisture > fat > carbohydrate > protein. There is a positive correlation between fission gamma absorbed dose rate and the weight of ash, water and fat, while a negative correlation with carbohydrate and protein. However, the linear relationship between each component and the absorbed dose rate of induced gamma is not significant, this needs to be identified by further researches. Based on the results of the present study, we conclude that the neutron absorbed dose can be calculated without taking into account the fat composition of bean crop seeds (except for soybean seeds) in the process of mutation breeding induced by radiation. In special cases where the accuracy requirement of the dose rate is not high, it is possible to use protein instead of legume crop seeds for neutron absorption dose calculations.

Chiral selection rules for multi-photon processes in two-dimensional honeycomb materials.

We examine the chirality-dependent optical selection rules in two-dimensional monolayer materials with honeycomb lattices, and, based on symmetry argument, we generalize these rules to multi-photon transitions of arbitrary orders. We also present the phase relations between incident and outgoing photons in such processes. The results agree nicely with our experimental observations of second- and third-harmonic generation. In particular, we demonstrate that the phase relation of chiral second-harmonic generation can serve as a handy tool for imaging domains and domain boundaries of these monolayers. Our results can benefit future studies on chirality-related optical phenomena and opto-electronic applications of such materials.

Stacking symmetry governed second harmonic generation in graphene trilayers.

Crystal symmetry plays a central role in governing a wide range of fundamental physical phenomena. One example is nonlinear optical second harmonic generation (SHG), which requires inversion symmetry breaking. We report a unique stacking-induced SHG in graphene trilayers, whose individual monolayer sheet is centrosymmetric. Depending on layer stacking sequence, we observe a strong optical SHG in a Bernal ABA-stacked non-centrosymmetric trilayer, while it vanishes in a rhombohedral ABC-stacked one, which preserves inversion symmetry. This highly contrasting SHG due to the distinct stacking symmetry enables us to map out the ABA and ABC crystal domains in an otherwise homogeneous graphene trilayer. The extracted second-order nonlinear susceptibility of the ABA trilayer is surprisingly large, comparable to the best known two-dimensional semiconductors enhanced by excitonic resonance. Our results reveal a novel stacking order-induced nonlinear optical effect, as well as unleash the opportunity for studying intriguing physical phenomena predicted for stacking-dependent ABA and ABC graphene trilayers.

Combining reverse-transcription multiplex PCR and microfluidic electrophoresis to simultaneously detect seven mosquito-transmitted zoonotic encephalomyelitis viruses.

Several mosquito-transmitted viruses are causative agents for zoonotic encephalomyelitis. Rapid identification of these viruses in mosquito populations is an effective method for surveying these diseases. To detect multiple mosquito-transmitted viral agents, including West Nile virus, Saint Louis encephalitis virus, Venezuelan equine encephalomyelitis virus, Western equine encephalomyelitis virus, Eastern equine encephalomyelitis virus, Highlands J virus and Japanese encephalitis virus, an assay using multiplex reverse-transcription PCR combined with microfluidic electrophoresis was developed and evaluated. Tailed nested primers were used in the assay to amplify specific viral genomic segments, and products with specific length were further analyzed by using a microfluidic electrophoresis chip. The assay exhibited good specificity and analytical sensitivity (10(2) copies/µL). This technology can be helpful in the quarantine and surveillance of exotic encephalomyelitis viruses which are transmitted by mosquitoes.

Structures of potassium calix[4]arene crown ether inclusion complexes and application in polymerization of rac-lactide.

Reaction of 1,3-dipropoxy-p-tert-butyl-calix[4]arene (L(1)H2) with KN(SiMe3)2 afforded a one-dimensional (1D) chain complex [K2L(1)]n (1). Upon reaction with 1 equivalent 18-crown-6, complex 1 can convert to complex [K2(18-crown-6)L(1)] (2) which possesses a sandwich structure. Treatment of two calix[4]arene-crown ligands of 1,3-dihydroxy-p-tert-butyl-calix[4]arene-crown-5 (L(2)H2) and 1,2-dihydroxy-p-tert-butyl-calix[4]arene-crown-5 (L(3)H2) with KN(SiMe3)2 gave the dinuclear complex [K2L(2)] (3) and the mononuclear complex [K(THF)L(3)H] (4), respectively. Complexes 1-4 were all characterized by single-crystal X-ray diffraction techniques. The variable temperature (1)H NMR spectrum indicates there is a quick rotation equilibrium of the two phenoxy groups in complex 3. In addition, complexes 1-4 have been tested for the ring-opening polymerization (ROP) of rac-lactide and the results showed that complexes 2 and 3 are highly active for the ROP of rac-lactide. The obtained polymers displayed low dispersity values (D) and the molecular weights are close to the calculated ones. Furthermore, complexes 2 and 3 show moderate isoselectivities of Pm = 0.67 and Pm = 0.73, respectively.

Bulky metallocavitands with a chiral cavity constructed by aluminum and magnesium atrane-likes: enantioselective recognition and separation of racemic alcohols.

Seven new type metallocavitand complexes 1­7 were synthesized via the self-assembly of aluminum and magnesium atrane-likes. The recognition of R-2-butanol from racemic 2-butanol can be achieved in the chiral cavity of metallocavitand complex 5. The crystal structure of complex 5 showed that the enantioselectivity of the center cavity for the inclusion of two 2-butanol molecules is higher than that of the groups at the outer rim, which indicates that the size-limited cavity is more sensitive to the chirality of 2-butanol. Furthermore, desorption of R-2-butanol is successful through vacuumization which afforded complex 6 and gives R-2-butanol with an enantiomeric excess (ee) value of 53(±1)%. The reaction of enantiopure H3L2, MgnBu2, and racemic 1-phenylethanol afforded complex 7. The structure of complex 7 showed that the center cavity was occupied by three H2O molecules and one molecular R-1-phenylethanol suspended in the outer rim of the metallocavitand via a hydrogen bond, which indicated that 1-phenylethanol is too bulky for the size-limited cavity. Because a certain amount of racemic 1-phenylethanol is also co-crystallized in the unit cell, the final separated 1-phenylethanol has an ee value of 33(±1)%. The host­guest mechanism for the separation is clearly determined through X-ray crystal structural analysis.

Valley and band structure engineering of folded MoS(2) bilayers.

Artificial structures made of stacked two-dimensional crystals have recently been the focus of intense research activity. As in twisted or stacked graphene layers, these structures can show unusual behaviours and new phenomena. Among the various layered compounds that can be exfoliated, transition-metal dichalcogenides exhibit interesting properties governed by their structural symmetry and interlayer coupling, which are highly susceptible to stacking. Here, we obtain-by folding exfoliated MoS2 monolayers-MoS2 bilayers with different stacking orders, as monitored by second harmonic generation and photoluminescence. Appropriate folding can break the inversion symmetry and suppress interlayer hopping, evoking strong valley and spin polarizations that are not achieved in natural MoS2 bilayers of Bernal stacking. It can also enlarge the indirect bandgap by more than 100 meV through a decrease in the interlayer coupling. Our work provides an effective and versatile means to engineer transition-metal dichalcogenide materials with desirable electronic and optical properties.

Development of loop-mediated isothermal amplification assay for specific and rapid detection of differential goat pox virus and sheep pox virus.

BACKGROUND: Capripox viruses are economically important pathogens in goat and sheep producing areas of the world, with specific focus on goat pox virus (GTPV), sheep pox virus (SPPV) and the Lumpy Skin Disease virus (LSDV). Clinically, sheep pox and goat pox have the same symptoms and cannot be distinguished serologically. This presents a real need for a rapid, inexpensive, and easy to operate and maintain genotyping tool to facilitate accurate disease diagnosis and surveillance for better management of Capripox outbreaks. RESULTS: A LAMP method was developed for the specific differential detection of GTPV and SPPV using three sets of LAMP primers designed on the basis of ITR sequences. Reactions were performed at 62°C for either 45 or 60 min, and specificity confirmed by successful differential detection of several GTPV and SPPV isolates. No cross reactivity with Orf virus, foot-and-mouth disease virus (FMDV), A. marginale Lushi isolate, Mycoplasma mycoides subsp. capri, Chlamydophila psittaci, Theileria ovis, T. luwenshuni, T. uilenbergi or Babesia sp was noted. RFLP-PCR analysis of 135 preserved epidemic materials revealed 48 samples infected with goat pox and 87 infected with sheep pox, with LAMP test results showing a positive detection for all samples. When utilizing GTPV and SPPV genomic DNA, the universal LAMP primers (GSPV) and GTPV LAMP primers displayed a 100% detection rate; while the SPPV LAMP detection rate was 98.8%, consistent with the laboratory tested results. CONCLUSIONS: In summary, the three sets of LAMP primers when combined provide an analytically robust method able to fully distinguish between GTPV and SPPV. The presented LAMP method provides a specific, sensitive and rapid diagnostic tool for the distinction of GTPV and SPPV infections, with the potential to be standardized as a detection method for Capripox viruses in endemic areas.

Preparation and evaluation of a mixed-bed immunoaffinity column for selective purification of sixteen sulfonamides in pork muscle.

This paper describes the preparation of a novel mixed-bed immunoaffinity chromatography (IAC) column by coupling four monoclonal antibodies against different sulfonamides (SAs) to Sepharose 4B. The IAC column can be used to simultaneously extract and purify 16 SAs in pork muscle. The dynamic column capacities for all SAs in mixed standard solution were between 312 and 479 ng/mL gel. After simple extraction and IAC cleanup, the sample solution can be directly injected for liquid chromatography-ultraviolet analysis. The recoveries of SAs from spiked samples at levels of 25, 50 and 100 µg/kg ranged from 83.3 to 103.1% with variation coefficient less than 8.6%. The comparison of IAC with liquid-liquid extraction and solid phase extraction indicated that IAC has better purification effect and needs less organic solution than conventional methods, thus it would be an ideal method for selective purification of SAs in pork muscle.