Novel nanoporous Gl-POSS-branched polymers based on the macroinitiator of anionic nature, 2,4-toluene diisocyanate, and octaglycidyl polyhedral oligomeric silsesquioxane (Gl-POSS) were obtained as gas separation membranes. The synthesis of polymers was carried out using various loads of Gl-POSS. It was found that the main reaction proceeding with 2,4-toluene diisocyanate is the polyaddition, accompanied by the isocyanate groups opening of the carbonyl part. This unusual opening of isocyanate groups leads to the formation of coplanar acetal nature polyisocyanates (O-polyisocyanate). The terminal O-polyisocyanate links initiate the subsequent opening of the epoxide rings in Gl-POSS. As a result, Gl-POSS serves as a hard and bulky branching agent and creates the specific framing supramolecular structure, which leads to the formation of nanopores in the polymer, where the flexible polyether components are located inside the cavities. Thermal, mechanical, physical, and chemical properties of the obtained polymers were studied at various Gl-POSS contents in the polymer matrix. It was found that these polymers show high selectivity of gas transport properties for pure ammonia relative to nitrogen and hydrogen at ambient temperature. Measurements showed that the gas permeability coefficients and the values of ideal selectivity were in a non-additive dependence to the Gl-POSS content.  Throughout recent decades, greater attention has been turned toward the use of polymeric gas separation membranes in separation and purification processes. This is largely owing to their potential energy efficiency and minimal ecological impact as compared to traditional, but potentially dangerous, distillation, crystallization, and sorption methods ,2,3. DesConversely, gas permeation through rubbery polymers is due to weak interactions between the macromolecules. As a result, the permeability is higher as compared to glassy polymers, but the selectivity is significantly diminished ,2,3,4,5.Another possible improvement of gas separation membrane properties  is active transport, especially in the case of the separation of nonpolar gases, and active penetrants like carbon dioxide ,20,21, h−4 cm3/cm2 sec cm Hg) n[CH2(CH3)CH2O]m[CH2CH2O]nK, where n ≈ 14 and m ≈ 48, molecular weight 4200, content of potassium alcoholate groups is 10.9 wt% from the total the number of functional groups, was purchased from PJSC Nizhnekamskneftekhim . 2,4-toluene diisocyanate 98% (TDI) was purchased from Sigma-Aldrich. Octaglycidyl polyhedral oligomeric silsesquioxane (Gl-POSS) was purchased from Hybrid Plastics . Gl-POSS is unstable over time and thus was closed tightly and stored in a cold dry place at 283 K. The reaction media for the synthesis of block-copolymers was ethyl acetate. PPEG was additionally dried at reduced pressure (approximately 0.07 kPa) and at an elevated temperature of 353 K down to a 0.01 wt% moisture concentration. Block-copolymer of propylene oxide with ethylene oxide (PPEG) with formula HO (g/eq) and  groups (E/K) was taken into account, when calculating the content of Gl-POSS. The reaction was carried out in ethyl acetate at 25 °C in a flask equipped with a reflux condenser. The polymerization process proceeded with constant stirring using a magnetic stirrer. The flask contained PPEG (1 g) and ethyl acetate (4.86 g). The calculated amount of Gl-POSS as a curing agent was added to PPEG solution and under constant stirring. The reaction mass was mixed at a given temperature until the complete dissolution of Gl-POSS took place. After this, TDI (0.62 g) at a molar ratio of [PPEG]:[TDI] = 1:15 was introduced. The Gl-POSS content varied in the range of 0.0 to 20 wt%, and depending on this, the polymer was designated as POI-0, POI-Gl-POSS-0.1, POI-Gl-POSS-0.4, POI-Gl-POSS-0.5, POI-Gl-POSS-1, POI-Gl-POSS-2, POI-Gl-POSS-5, POI-Gl-POSS-8, POI-Gl-POSS-10, POI-Gl-POSS-15, and POI-Gl-POSS-20. The resulting solution of the polymer-forming system was cast in a Petri dish and cured at ambient temperature for 72 h. The ratio of [~OAll samples were seasoned at room temperature at least 3 weeks for removing the solvent residuals before performing mechanical, physical, and gas transport tests.Tensile stress–strain measurements were obtained from the film samples of size 40 mm × 15 mm with Universal Testing Machine Inspekt mini  at 293 ± 2 K, 1 kN. The crosshead speed was set at 50 mm/min and the test continued until sample failure. A minimum of five tests were analyzed for each sample and the average values were reported. −1, and the number of scans was 60.The Fourier transform infrared (FTIR) spectra of the products were recorded on an InfraLUM FT 08 Fourier transform spectrometer  using the attenuated total reflection technique. The spectral resolution was 2 cmThe thermomechanical curves of polymer samples were obtained using a TMA 402 F  thermomechanical analyzer in the compression mode. The thickness of the sample was 2 mm, the rate of heating was 3 K/min from room temperature to 540 K in static mode, and the load was 2 N. The MLT curves of polymer samples were taken using the dynamic mechanical analyzer DMA 242  in the mode of the oscillating load. The force and stress–stain correspondence were calibrated using a standard mass. The thickness of the sample was 2 mm. Viscoelastic properties were measured under nitrogen. The samples were heated from room temperature to 540 K at the rate of 3 K/min and a frequency of 1 Hz. The mechanical loss tangent was defined as the ratio of the viscosity modulus G″ to the elasticity modulus G’.Thermal gravimetric analysis (TGA) was performed using a STA-600 TGA–DTA combined thermal analyzer . The samples (0.1 g) were loaded in alumina pans and heated from 297 to 870 K at a rate of 5 K/min in a nitrogen atmosphere. The temperature dependence of the dielectric loss tangent (DLT) of polymer samples (thickness of 0.5–0.7 mm) was registered in the temperature range from 130 to 370 K at a frequency of 1 kHz. A facility consisted of the measuring cell that was placed in a Dewar vessel filled with nitrogen and to which an E7-20 RLC-meter and a B7-78 universal voltmeter functioning as a precision thermometer were connected.An atomic force microscope  was used to reveal the morphology of the samples. The 250–350 kHz cantilevers  with silicone tips (tip curvature radius is of 10–13 nm) were used in all measurements. The microscopic images were obtained with a 512 × 512 resolution. The scanning rate was 1 Hz. Samples were fixed on a 12-mm metal SPM specimen discs using adhesive carbon tabs .3) to the surface of the membrane using a microsyringe. Adjust the position of the holder to obtain the sharpest image of the drop and plate. The parameters of the height h and the diameter d of the droplet are determined using an eyepiece micrometer. The drop is positioned so that it is located in the field of the eyepiece view between divisions 0 and 10. The image of a drop on the membrane is obtained by an inverted image. To measure the diameter of the drop base, the eyepiece-micrometer droplets rotate and move its crosshair vertically and combine it with the image of the plate (with the drop interface—the surface of the plate). The boundaries of the left and right droplet angle are marked in units of the scale division, n1 and n2, and then the parameter d is calculated by the difference n2 − n1. The drop height is determined similarly by turning the scale 90°. The angle of capillarity is a characteristic of the hydrophilicity (hydrophobicity) of the membrane surface. It is defined as the angle between the tangent drawn to the surface of the wetting liquid and the wetted surface of the solid, while always counting from the tangent to the side of the liquid phase. The tangent is drawn through the point of the three-phase contact: Solid phase (membrane), liquid (distilled water), and gas (air). Measurements were performed using an eyepiece micrometer. The measuring cell is mounted on a holder table. The test membrane is placed on a stand in the measuring cell and the illuminator lamp is turned on. Then, 20 mL of the test liquid is poured into the cuvette so that the liquid level is equal to half the height of the stand in the measuring cell. A drop of this liquid is carefully applied  was operated in the standard pressure range up to 35 bar. Porefil  was used as the wetting liquid with the lowest surface tension available on the market equal to 16 dyne/cm. The sample holder diameter was 25 mm, and data acquisition and analysis were carried out using LabViewThe pure component permeances through the obtained POI-Gl-POSS membrane were measured according to the Daynes–Barrer technique ,60,61,62−5 Pa. The membrane area was 2.5 × 10−4 m2. The feed- and permeate-side pressures were monitored by high-precision pressure sensors WIKA S-10 0-16 bar  having an accuracy of 0.5% of span (for the feed side), and MKS Baratron 750B 0–20 torr pressure transducer  with an accuracy of 1% of reading (for the permeate side). Constant feed pressure was maintained at 110 kPa; the permeate pressure variation was recorded with a sampling rate of 10 ms. Each single-gas test was repeated at least three times at least 24 h apart before each run. As a permeation cell, a radial membrane module . After ammonia analysis, the permeability of hydrogen and nitrogen was tested again to show the chemical resistance of polymer. All experiments were carried out following safety precautions, which are specified in ISO/TC 58/SC 4 and ISO/TC 161. After the investigation of ammonia gas transport properties, the permeability coefficients of hydrogen and nitrogen did not change. The single gas permeability values of hydrogen, nitrogen, and ammonia through each sample were measured for 3 gases  of mol m m−2s−1Pa. The permeability coefficient was performed in Barrer (1 Barrer = 3.348 × 10−16 mol m m−2 s−1 Pa−1 = 10−10 cm3 (STP) cm cm−2 s−1 cm Hg−1). Moreover, the Daynes–Barrer technique ,60,61,623/N2, NH3/H2. The choice of the gas pairs is stipulated by the necessity of the ammonia separation in the Haber Bosch process, which is an artificial nitrogen fixation process and the main industrial procedure for the production of ammonia today.After permeability measurements, the ideal selectivity calculated as the ratio of the fast over the slow permeability coefficients for the following pair of gases: NHThe permeability coefficient relative error for a single test is less than 5%; consequently, the selectivity relative error is 7.1%. The relative error of the measurement series was usually less than 15%. Taking into account the thickness measurement relative error of 2.4%, the diffusion coefficient relative error is 5.5% and the sorption coefficient relative error equals 7.5%.Samples thickness was measured using the micrometer  with a range of 0–25 mm with an error of ±5 µm. The polymeric film was covered with a flat glass plate of a known thickness to prevent direct contact of the measuring tip of the micrometer with the soft film. The thickness of the samples was 287.5, 298, 210, 234, 267, 314, and 283 µm in order of the Gl-POSS content increase in the samples.α1 1.5406 Å). Room-temperature data were collected in the reflection mode. Samples in the form of rectangular fragments cut from the initial films 2.5 cm × 2.5 cm in size were placed in a standard sample holder, which was kept spinning (15 rpm) throughout the data collection. The diffraction patterns were recorded in the range of scattering angles 2θ 1°–70°, step 0.008°, with a step time 0.1–0.5 s.X-ray powder diffraction (XRPD) measurements were performed on a Bruker D8 Advance diffractometer equipped with a Vario attachment and Vantec linear PSD, using Cu radiation  monochromated by a curved Johansson monochromator (λ Cu K−1 < s < 0.34 Å−1 (s = (4π/λ)sin(θ)). Scattering patterns were obtained for the samples at 23 °C in an evacuated chamber. The measurements were performed in transition mode with the use of samples in the form of films of a uniform thickness and 4 × 8 mm2 dimension. Samples were mounted on a standard sample holder and placed in an evacuated diffractometer chamber. For each sample, several experiments were performed, allowing control of the quality of the experiments. The 2-D scattering patterns were integrated using the SAXS program package [Small angle X-ray scattering (SAXS) data for samples were collected with the Bruker AXS Nanostar SAXS system using CuKα (λ 1.5418Å) radiation from a 2.2 kW X-ray tube  coupled with Gobbel mirrors optics and a HiStar 2D area detector. The beam was collimated using three pinholes with apertures of 800, 450, and 700 μm. The sample–detector distance was 64.2 cm and was controlled using an AgBh calibration sample. The range of available values of scattering angles was 0.1° < 2θ < 4.8°, which corresponds to the values of the wave vector equal to 0.007 Å package . The dat package  and SASV package  program The opening of isocyanate groups of TDI initiated by a macroinitiator (PPEG) can proceed both at the C=O and at the N=C component  53,54,6,654,69, It was shown  that theThe main process in the interaction is the polyaddition of TDI accompanied by the isocyanate groups’ opening of the carbonyl part and opening of the epoxide ring initiated by a terminal O-polyisocyanate link as shown by the proposed mechanism in −1 due to the stretching vibrations of C=O bond of the isocyanurates. A shoulder at 1730 cm−1 is seen, characteristic for the C=O bond in the urethane group. The chemical structures of POI-0 and POI-Gl-POSS were confirmed by FTIR analysis. −1 and the comparability of the band intensities at 1710 and 1730 cm−1, it can be concluded that only a small part of the isocyanate groups participates in isocyanurates and urethane formation processes  on the TMA curves due to the thermal effect on the segmental mobility of the flexible amphiphilic component.The content of Gl-POSS has the greatest influence on the high–temperature ranges of relaxation processes. At 503 K, strain changes do not exceed 20–40%. After 513 K, deformation of the polymer is determined by the mass loss as a result of the thermal degradation. It should be noted that under the mechanical load, the sample obtained at 10 wt% of Gl-POSS has the highest thermal resistance and reaches 55% strain at 623 K.The results indicate that the O-polyisocyanate blocks are the key element of the supramolecular organization of the studied polymers. Owing to this functionality, the domains are stable at high temperatures. Such stability of the domain structures suggests that urea, formed by the reaction of the isocyanate groups of the ortho-position in TDI, forms bridges between adjacent O-polyisocyanate units, leading to their stabilization and segregation due to both chemical and physical binding according to The POI-0 and POI-Gl-POSS composites were characterized by the dielectric loss tangent (DLT) and mechanical loss tangent (MLT) to study their bulk properties. The use of Gl-POSS and the increase of its content amplify the resistance of POI to thermomechanical effects by forming framing structures. Due to the existence of O-polyisocyanates as a part of the rigid branched structure, the polymer matrix has spatially different architecture elements of rigid components. This fact is reflected in the temperature dependences of the MLT for POI-Gl-POSS. The peak of the temperature dependence of DLT at 246 K is most likely by the α-transition of the polyoxyethylene block. For POI-Gl-POSS-2 and POI-Gl-POSS-5, the α-transition at 226 K becomes less pronounced, while at the range of POI-Gl-POSS from 2 to 5, the region of the α-transition at 246 K is observed as a well-separated peak. This suggests that under such conditions, the polyoxyethylene component is directly adjoined to the hard O-polyisocyanate unit and involved in the formation of spatial units of the polymer network and loses its ability to isolate. For POI-Gl-POSS-10, POI-Gl-POSS-15, and POI-Gl-POSS-20, the α-transition at 226÷231 K is more expressed and broad. The presence of only one peak in the α-transition region is caused by irregularity of the skeleton supramolecular structure at such high contents of Gl-POSS. As a result, the excellence of the microphase separation of polyoxypropylene and polyoxyethylene units is lost.The static mechanical properties of the polymers were estimated by tensile tests at ambient temperature. The samples used for the tensile test were obtained in three areas of the Gl-POSS content  . Gl-POSSWhen the content of Gl-POSS is in the range from 1 wt% to 8 wt%, the obtained samples are high-modulus polymers and are not typical elastomers. The stress values at 7–8 wt% tensile strain depend on the content of Gl-POSS, and reached 60 MPa at 2 wt% of Gl-POSS. At less than 1 wt% and more than 8 wt% of Gl-POSS, the stress value at a tensile strain of 7%–8% drops significantly. The geometric dimensions of the samples returned to its original size within 2–5 min after the applied stress and the subsequent break. Thus, when the content of Gl-POSS is in the range from 1 wt% to 8 wt%, the tensile strength gets many times higher, accompanied by a sharp decrease in the elongation at breaking stress. The results were explained as follows. Gl-POSS is involved in the chemical reactions initiated by the active centers at the end of the hard O-polyisocyanate blocks. As a result, Gl-POSS serves as a hard and bulky branching agent. In the end, the formed O-polyisocyanate blocks make an association around Gl-POSS. In the case of the optimal Gl-POSS content, branched geometry is seen, and the domains of the O-polyisocyanate blocks create a rigid frame, having an inner volume greater than the volume of the flexible component, which is 50 wt%. As a result, the flexible component covers the surface of the geometrically aligned domains of hard segments, while the internal cavity of the frame is empty. A feature of such a supramolecular architecture is that the structure of the polymer becomes framing, and thereby flexible polyether components are located inside the cavities, preventing flexing in the process of uniaxial tension. Under these conditions, the polyoxypropylene component of the macroinitiator forms its own microphase outside the segregation of hard segments and Gl-POSS domains, while the polyoxyethylene component of the macroinitiator exists inside the area of formation of the spatial branched structures. The content of Gl-POSS below 1 wt% and higher than 8 wt% destroys the strict geometry of the frame. This results in the loss of the necessary geometry for the supramolecular structures and, thus, in deterioration of the physical and mechanical characteristics of the polymer with the Gl-POSS content below 1% and higher than 8% . In thisThe X-ray powder diffraction data for the polymer samples with different Gl-POSS contents indicate the absence of crystalline phases in them and the amorphous state of the samples, which suggests that the association of POSS molecules into nanocrystallites does not occur.Small-angle X-ray scattering of POI-Gl-POSS polymers of different compositions indicates microphase separation of the rigid and flexible components in these systems and the formation of ordered supramolecular systems of the paracrystalline type. In this case, the two-dimensional patterns correspond to the scattering from isotropic systems, as evidenced by a uniform intensity distribution around the primary X-ray beam . For comd in the studied samples (the distance between the centers of the domains) lie in the range of 85.3–82.0 Å. For the POI-Gl-POSS-5 polymer, the parameter d is noticeably smaller and equal to 79.1 Å. An estimate of the average sizes of the areas of paracrystalline ordering (Long range order) leads to a value of 273.1 Å for polymer POI-Gl-POSS-5, and to a range of 330.2–371.8 Å for the remaining samples.The interference peaks indicate the structural ordering of the samples, i.e., about the availability of domains and their ordered arrangement in the volume of samples. The parameters of this ordering are fairly close for all the samples studied. The parameters of the paracrystalline lattice A characteristic feature of the scattering pattern of sample with the highest Gl-POSS content (POI-Gl-POSS-15) is intense scattering in the region of the smallest scattering angles d, which Taking into account the Babinet principle , with eqThree 2-D SAXS patterns are shown for each of the samples, which correspond to the initial state of the samples, the swollen state, and after 2 days of keeping the samples under room conditions. It can be noted that for the sample with the highest Gl-POSS content (POI-Gl-POSS-15), an irreversible disappearance of scattering is observed in the region of the smallest angles, which confirms to some extent the assumption of the presence of voids in this sample .An analysis of the SAXS data of the samples obtained before swelling, after swelling in water and after settling in room conditions allowed us to confirm the conclusions based on the results of the physical and mechanical tests. So, typical for the samples is the skeleton of the supramolecular structure due to the presence of domains and bulk fragments connecting them. The absorption of water by the samples under study was carried out into voids and did not lead to disruption of the frame supramolecular structure due to the stiffness and the predominant contribution to its formation of chemical rather than intermolecular bonds.2, H2) and ammonia. Changes in the macromolecular and supramolecular structure are reflected in the permeability values of the inert gases and ammonia. The obtained values for the POI-Gl-POSS membranes with different Gl-POSS contents at ambient temperature are presented in 2 and NH3 molecules’ kinetic diameters, which are 2.89 and 2.6 Å [2 diffusion coefficients increase with an increase of the POI-Gl-POSS load in the samples; meanwhile, the NH3 diffusion coefficient remains the same (about of 3.8 m2 s−1), except POI-Gl-POSS 1 wt% with 5.9 m2 s−1. Nevertheless, the high ammonia permeability rate was mainly determined by its sorption rate in the studied samples, the value of which increases with the POI-Gl-POSS loading rate. A further increase in the POI-Gl-POSS content leads to a decrease in both the sorption and, as following, the permeability. The H2 sorption coefficients are comparatively low, due to the absence of an interaction with the membrane material. In light of the above, the high selectivity for the NH3/H2 system is determined mainly by the samples’ sorption ability. As for nitrogen, its diffusion coefficient should be smaller, because the kinetic diameter of the nitrogen molecule is 3.64 ÅThe obtained diffusion coefficients are in good agreement with the Hnd 2.6 Å , respectThe difference between the permeability of the control sample and the samples obtained at a very low Gl-POSS content (0.1–1.0 wt%) is a result of its participation in the reaction process, and shows a significant impact of the cooperative effects on the formation of the supramolecular structure of POI-Gl-POSS. Nitrogen molecules hardly pass through the polymers POI-Gl-POSS-0.1, POI-Gl-POSS-0.5, and POI-Gl-POSS-1.0, and the ammonia permeability increases with the growing Gl-POSS content.The most likely reason is that in this case, the frame supramolecular structure is fragmentary, making the contact between the pores difficult. The frame pieces are alternated with the flexible chain component, which forms its own microphase outside the segregation of the hard segment domains and Gl-POSS. As a result, the supramolecular alignments alternate within the polymer, so that they form voids with the layers of the flexible component.2 and N2 molecules do not penetrate through the flexible chain, and this explains their very low permeability as shown in The non-polar HAccording to this study, the regular frame supramolecular structure is formed at 2–8 wt% of Gl-POSS. The inert gases’ permeability increases, but the ammonia permeability value slightly reduces in this range of the Gl-POSS content. The most likely reason of the revealed behavior is the alignment of the voids in a system of interconnected channels. Thus, the results indicate that ammonia is dissolved in PPEG. Hence, the supramolecular structural features of POI-Gl-POSS polymeric membranes introduced in this study result in a significant improvement of the separation efficiency of the ammonia-containing gas mixtures.The performance, i.e., permeability, must be improved to be competitive with commercially available gas separation membranes. 3/N2 and NH3/H2 systems. Another one, among the well-studied polymers, poly(vinyltrimethylsilane), was characterized by an ammonia permeability of 830 Barrer and selectivity of 7.5 and 4.1 with regard to nitrogen and hydrogen, respectively [3 of 360,000 Barrer [3/H2 system of 50. Unfortunately, there is no data on nitrogen in this paper. The commercially available membrane Nafion 117 (Poly(perfluorosulfonate)) [3 permeability of 323,070 Barrer and very high selectivity for both NH3/N2 and NH3/H2 pairs of 702 and 452. It was shown in a number of studies ,73,74,75ectively . The pol0 Barrer  and quitfonate))  demonstr3/N2 and NH3/H2 systems. This fact may lead to the use of these membranes in an ammonia separation plant application as shown in The obtained results for POI-Gl-POSS membranes seem to be promising due to their high selectivity values for NHIn summary, this paper described a novel nanoporous Gl-POSS-branched polymer synthesized by polyaddition of 2,4-toluene diisocyanate with macroinitiator, and using octaglycidyl polyhedral oligomeric silsesquioxane as a bulk branch node. It was found that the main process in the interaction is the polyaddition of 2,4-toluene diisocyanate accompanied by the isocyanate groups’ opening of the carbonyl part. The results suggest that the participation of Gl-POSS in the reaction with reactive terminal O-polyisocyanate links leads to the formation of the frame supramolecular structure. In the Gl-POSS content of 1–8 wt%, the supramolecular structure is the most ordered, and it fully reveals the segregated activity of the rigid component. Under these conditions, the polyoxypropylene component of the macroinitiator forms its own microphase outside the segregation of hard segments and Gl-POSS domains, while the polyoxyethylene component of the macroinitiator exists inside the area of the formation of the spatial branched structures. As a result, the polyoxypropylene component covers the surface of the geometrically aligned domains of hard segments, while the internal cavity of the frame is empty.It was shown that the largest values of ideal selectivity towards the ammonia-gas system were achieved at the content of Gl-POSS in the amount of 0.5–1 wt% and higher than 2–8 wt% in the polymer. It has been suggested that the combination of high permeability with high selectivity for ammonia molecules is the result of alternating relatively large voids with the layers of block copolymers of ethylene oxide and propylene oxide, through which ammonia dissolves.


We design InGaAs/AlInAs SL APDs with three different SL periods  to achieve the same composition as Al0.4Ga0.07In0.53As quaternary random alloy (RA). The simulated results of an RA and the three SLs predict that the SLs have lower k-values than the RA because the electrons can readily reach their threshold energy for impact ionization while the holes experience the multiple valence minibands scattering. The shorter period of SL shows the lower k-value. To support the theoretical prediction, the designed 6 ML and 8 ML SLs are experimentally demonstrated. The 8 ML SL shows k-value of 0.22, which is lower than the k-value of the RA. The 6 ML SL exhibits even lower k-value than the 8 ML SL, indicating that the shorter period of the SL, the lower k-value as predicted. This work is a theoretical modeling and experimental demonstration of engineering avalanche characteristics in InGaAs/AlInAs SLs and would assist one to design the SLs with improved performance for various SWIR APD application.We report on engineering impact ionization characteristics of In APDs are preferred for these applications, relative to conventional p-i-n PDs, because of the internal multiplication gain. This gain significantly improves the sensitivity of a sensing and imaging system when the circuit noise limits the signal to noise ratio (SNR). Therefore, APDs allow one to detect a lower signal magnitude coming through any harsh environment that degrades the initial intensity of the input signal.Avalanche photodiodes (APDs) have been widely used for various short-wavelength infrared  applications such as optical communicationM), the dark current (Id), and the excess noise factor (F(M)). When a high electric field is applied to the materials, carriers (electrons and holes) can gain enough energy to impact ionize, resulting in M. However, this high electric field gives rise to large tunneling dark current due to relatively narrow bandgap and high excess noise caused by a stochastic process of multiplication events in the materials. This problem can be mitigated by using separate, absorption, charge, and multiplication (SACM) APDs, where a high electric field is located at a wide bandgap multiplication layer (M-layer) while a low electric field exists in a narrow bandgap absorption layer. To prevent strain-induced defects that reduce the device performance, SACM APDs need to be made from material layers that are lattice-matched to the host substrate. InP substrates are commonly used for commercial APD technologies because they are reasonably cheap and commercially available up to 4″ in diameter. Since good absorbers such as In0.53Ga0.47As (1550 nm)5 and In0.53Ga0.47As/GaAs0.51Sb0.49 (2000 nm)6 on InP substrates are already well-developed, they can easily be combined with wide bandgap materials used for the M-layer such as InP7, Al0.48In0.52As8, AlxGa1−xAs0.56Sb0.449 and AlAs0.56Sb0.4410, potentially realizing high-performance SACM APDs.There is interest in APDs operating at 1500 nm or 2000 nm for LIDAR active sensing and imaging applications. The three critical characteristics of an APD are the multiplication gain (α) and holes (β). The excess noise can be quantified as F(M), and is estimated by McIntyre’s local field theory11, F(M) = kM + (1 − k) [2–1/M]. Here, k is the ratio of α and β impact ionization coefficients, specifically k = β/α when β > α and k = α/β when β < α, by convention. It should be noted that when k-value is low, F(M) increases slowly as M increases. Thus, a low k-value is desired to make an APD highly sensitive. A low k-value in an M-layer can be achieved simply by selecting a material system that has favorable α or β. For instance, Si has a k-value of < 0.0512, and InAs13 and HgCdTe14 have k-values of 0. There has been active research to find suitable materials for the M-layer that are lattice-matched to InP substrates. Tan and David et al. have developed thin AlxGa1−xAs0.56Sb0.44 APDs (hereafter AlGaAsSb) on InP substrates15. A 100 nm thick M-layer provided with a k-value of 0.1 with the assistance of dead space. However, an APD technology at least needs to be comparable to Si APDs, and the AlGaAsSb APDs cannot achieve such a low k-value with relatively thin layers. A way to attain the low k-value is to have an M-layer that is ~ 1500 nm thick16. Unfortunately, AlGaAsSb APDs cannot be randomly grown with such large thicknesses due to the existence of a large miscibility gap, causing phase separation during the growth.To minimize excess noise in an M-layer, there must be a large difference between the impact ionization coefficients for electrons  refers to half the lattice constant along the (100) direction for a zinc blende structure. We use the term SL in this paper to refer to periodic In0.53Ga0.47As/Al0.48In0.52As heterostructures, and specifically consider periods of three values . The limiting case of a 2 ML SL is a random alloy. The important point is that engineering the periodicity of the lattice has a dramatic impact on the band structure and the transport of carriers. Campbell and Bank et al. have demonstrated an 890 nm thick AlxIn1−xAsySb1−y SL grown on a GaSb substrate using four binary layers in a period as an alternative to the bulk random alloy (RA)17. Their Al0.7In0.3As0.3Sb0.7 APD showed a k-value of 0.01, which is comparable to Si. Thick AlAs0.56Sb0.44 APDs on InP substrates have also been successfully grown as AlAs/AlSb SLs for a 1550 nm thick M-layer. The SLs exhibited an extremely low k-value of 0.00516. To investigate the origin of lower k-value in a SL APD, AlAs/InAs SL  APDs were grown on an InP substrate. They achieved a lower k-value (k = 0.03) than an Al0.48In0.52As RA (k = 0.24)18. The origin was investigated in terms of electronic band structures of the SL and RA APDs. It was found that a mini gap exists in the hole bands of the SL which inhibits the holes from gaining enough energy to impact ionization while electrons can easily reach their threshold energy for impact ionization. This demonstrates that the advantage of using an SL for the M-layer is that its electronic structure can be engineered to realize a low k-value. In other words, one can modify material properties such as effective masses, band gaps (Eg), α, and β with various thicknesses of the alternating layers of the SLs19. For example, Grein, Ghosh and Krishna have demonstrated electron-APD and hole-APD with InAs/GaSb type-II SLs for mid-IR applications, specifically an extremely low F(M) of 0.8–1.2 and k ~ 021. This indicates that if the electronic structure of an SL is properly designed, one can favorably achieve a high-performance APD.Recently, the superlattice (SL) or digital alloy (DA) growth technique has opened up a new path to growing materials for APDs and has unlocked a new means for reducing the xGayIn1−x−yAs (hereafter AlGaInAs) quaternary alloy has proven to be a useful material system for optoelectronic devices. For APDs, AlGaInAs alloys have only been exploited for grading and charge layers in SACM APDs, since they can be designed with bandgaps intermediate between the absorber and multiplication layers10. This material has not been used solely as an M-layer since APDs with AlGaInAs RAs have a relatively high k-value of between 0.24 (Al0.48In0.52As)22 and 0.5 (In0.53Ga0.47As)23. A 1000 nm thick Al0.24Ga0.24In0.52As RA showed a k-value of ~ 0.524. However, In0.53Ga0.47As/Al0.48In0.52As (hereafter InGaAs/AlInAs) heterostructure APDs have demonstrated a promising k-value of 0.05 due to the larger conduction band offset than the valence band offset between Al0.48In0.52As and In0.47Ga0.53As25. Therefore, InGaAs/AlInAs SL may have the potential to achieve a reduced k-value. Also, an InGaAs/AlInAs SL facilitates more straightforward material growth of SACM APDs with an In0.53Ga0.47As absorber.The Al0.4Ga0.07In0.53As RA. The SL periods were chosen to be 4 ML, 6 ML, and 8 ML. As our first step, we computed the electronic band structures and impact ionization coefficients for those SL periods. These results were compared with the compositionally similar RA and analyzed in terms of avalanche threshold energies and scattering mechanism for electrons and holes. We experimentally demonstrate the SLs with periods of 6 ML and 8 ML and show the theoretical predictions are in good agreement with the experimental results.In this work, we discuss theoretical modeling and experimental demonstrations of a p-i-n configuration of InGaAs/AlInAs SLs as M-layer candidates. The thicknesses of constituent layers in the SL were designed to have the same average alloy composition as AlThe entire theoretical computations were done at 240 K while experimental demonstrations were performed at 300 K. The reason is that at low temperatures, band engineering features are more pronounced because thermal energies restrict carriers to relatively few minibands. The difference between 240 and 300 K will not create significant inconsistency between theory and experiment.19, which also contains the employed input parameters and is summarized here for completeness. The SL electronic band structures were calculated employing a 14-band SL K.p method26. It should be noted that the reliability of the SL K.p method becomes weaker as the wavevector deviates from the zone-center. Therefore, the SL K.p approach should be considered as the source of a physical explanation with the understanding that the quantitative predictions are approximate. The calculations use a heterostructure restricted basis formalism non-perturbatively employing fourteen bulk bands27. The heterostructure multiband Hamiltonian is then transformed into Fourier space and diagonalized. An envelope function formalism28 is used to obtain the zone-center SL states. States throughout the zone are then evaluated using the SL K.p method29. The highly non-parabolic band structures and momentum matrix elements computed employing the 14-band SL K.p method are used directly with a mesh spacing of 0.002 Å−1 as input for the evaluation of the impact ionization rates of hot holes and electrons. The designs of RA and the three SLs appear in Table Eg) extracted from the electronic structures of all four materials. Their computed electronic structures at 240 K appear in Fig. A realistic and reliable SL electronic band structure is the principal input to evaluate impact ionization rates. Our methodology follows that of ref.Ec) were engineered to promote the impact ionization of hot electrons, whereas valence minibands (Ev) were designed to suppress the ionization of hot holes. This is accomplished through choosing the SL period to permit electrons to impact ionize from the lowest conduction miniband, but holes must scatter among multiple valence minibands to reach their impact ionization threshold. Transport inhibited by zone folding reduces hole-initiated impact ionization while electrons undergo impact ionization similar to that of a bulk material. This can be seen from the band structure appearing in Fig. 0.4Ga0.07In0.53As RA has its lowest conduction, and the highest valence bands that cross line an energy gap above the conduction band minimum (upper dotted red line) and an energy gap below the valence band maximum (lower dotted red line), respectively. These lines represent minimum impact ionization threshold energies. This contrasts with the 4 ML SL where the lowest conduction miniband just crosses the line, suggesting the shortest period 4 ML SL is superior in terms of the electron to hole impact ionization coefficient ratio differing from one. The 6 ML and 8 ML SLs do not exhibit these desirable band-structure characteristics. However, they still show electron-initiated APD behavior due to their much heavier hole masses compared to the RA and multiple valence bands scattering for holes.The electronic band structures of the SLs were designed to optimize the electron-initiated to hole-initiated impact ionization coefficient ratio. Specifically, the conduction minibands  and set of minibands to yield the impact ionization rate as a function of hot carrier energy. Figure 0.4Ga0.07In0.53As RA and the 4 ML SL. It should be noted that the plots were obtained under weak field conditions. Both have threshold energies of electrons  and hole  nearly equal to the energy gap. However, somewhat promising, the electron-initiated rate of the 4 ML SL has a stronger onset than that of the RA, and the hole-initiated rate of the 4 ML SL is overall lower than that of the RA. This promotes electron-initiated impact ionization over that of holes. The 6 ML and 8 ML SLs were not examined here since they are seen as possessing electronic structure features intermediate between the extremes of the bulk alloy and 4 ML SL.The formalism used to compute the impact ionization rates follows that of ref.32. The k-value of the 4 ML SL is lower than that of the RA, and the difference of their k-value becomes much more significant at the lower electric field region, as shown in Fig. Figure In this section, we will experimentally demonstrate the APDs designed in previous section. The 4 ML SL is not considered here due to the limited equipment available for the difficulty of achieving low background doping. However, the 6 ML and 8 ML SLs are realized and appeared to be enough for the demonstration of the physical prediction discussed in section I. All measurements were performed at 300 K while computations were carried out at 240 K.33.Capacitance–voltage (C–V) measurements were taken at a probe station using specialized test structures. These circular test structures were blind (had no aperture) and had a diameter of 300 µm, much larger than the photodiodes. The large area increases the device capacitance, making it easier to measure. The ratio of the junction area and surface area is proportional to the device’s diameter, so larger devices minimize surface effects. Phase corrections and open circuit corrections were taken prior to measurementsnbg, was calculated to be < 1 16 cm−3, indicating low residual impurities in the SL materials.C-V data at room temperature is shown in Fig. M, we needed to determine the unity gain point of the 6 ML and 8 ML APDs. For a punch-through APD such as a SACM APD, it is not straightforward to determine the unity gain point because the curves of C–V and I–V under illumination are not saturated at a bias voltage. However, for a PIN APD the unity gain point was easily deduced due to the flat region of the I–V curve under illumination and the saturated region of the C–V curve. The devices are shown to be fully depleted at −10 V as shown in Fig. Current–voltage (I–V) characteristics were measured with a Keithley 2400 source meter. A 543 nm He–Ne laser was used as an optical source to illuminate the devices. To calculate M of the 6 ML SL approached 20, but was over 25 for the 8 ML SL. The catastrophic surface breakdown which destroyed the devices occurred at slightly different biases for each device, causing their maximum M to differ. Thus, the small difference in the maximum M measured between the 6 ML and 8 ML SLs is not a conclusive result.The I–V data for the 6 ML and 8 ML SLs are shown in Fig. F(M) of the 6 ML SL drops below F(M) = 2 , unlike the 8ML SL. This indicates that the gain is deterministic in nature. In order to understand the non-local effects, we undertook the modeling of the dead space for two different multiplication layer thicknesses (200 nm and 2000 nm) as shown by the dotted blue lines. The electron’s dead space is calculated by dividing the electron ionization threshold energy by the electric field34. The electron ionization coefficients for AlGaInAs are obtained from Tsuji et al24. This shows that non-local effects are occurring in the sample with 6 ML SL, and thus deterministic multiplication event. This needs further investigation, especially when the gain is increased. The current tolerance of the analyzer prevented F(M) measurements at higher gains, indicated in Fig. 11, the k-value of the 8 ML SL was a close match to ~ 0.22. This value is lower than what has been previously reported for Al0.48In0.52As RA and Al0.24Ga0.24In0.52As RA24, indicating growing this material as a digital alloy reduces the excess noise. The 6 ML SL data does not fit to McIntyre’s model but shows significantly lower excess noise than the 8 ML SL.The results of the excess noise measurement are shown in Fig. 0.4Ga0.07In0.53As RA and three InGaAs/AlInAs SLs  and briefly analyzed their electronic structures. The SLs were also experimentally realized. Here, more details on engineering impact ionization characteristics are discussed in terms of material and device perspectives in the SLs.We modeled one AlEg, as described in Table Eth for the three SLs as ~ Eg. Due to the SL periodicity, the 1st BZ edge of the SLs in the growth-axis direction becomes very short compared with the RA. The 1st BZ width of the SLs reduces as the period of the SL increases, as shown in Fig. Eth,e from the lowest conduction miniband while holes must scatter between multiple minibands before reaching their threshold. The effective masses of the electron (m0) are plotted in Fig. k values because the relatively light electrons can more readily reach their threshold energy than holes. The 6 ML and 8 ML SLs are less desirable for high-performance APDs than the 4 ML one because the electron energy at 1st BZ edge lies below Eth,e. However, we observed in Fig. k-value than the RA, although they do not satisfy the criteria of an optimal SL design. We here consider another design factor for the SLs, the presence of mini gaps. In Fig. Ec and the lower energy edge of Ev in the growth-direction . The mini gaps are important for carrier transport in the SLs because these energies will either provide hot carriers with a bridge to tunnel to or with an obstacle to prevent them from tunneling to another higher miniband35. The mini gaps are mostly determined by the band offset between two alternating layers and the strength of the interaction between adjacent energy states to perturb each other. A zero band offset either at conduction or valence band is highly preferred35. In the case of the SLs, the mini gaps are not so large that they do not prevent electrons and holes from tunneling to the higher band energies, thus reaching impact ionization threshold. At the large applied electric field, the tunneling probability even increases further37. We note that mini gaps can be a massive hindrance to the hole due to the larger k-value.Interestingly, all SLs have almost the same Eg can originate from three design factors; the 1st BZ edge along the growth direction, the effective masses, and the mini gaps, as discussed. Therefore, in order to make a high-performance SL, the selection of constituent materials and the thickness of their layers are very crucial. From the perspective of device design, we can take advantage of thick or thin M-layers. For a thin M-layer that is submicron, the impact ionization characteristics of carriers are highly influenced by the history of carrier transport due to the presence of the dead space, which is the distance where carriers need to travel before impact ionization38. In this situation, the probability density function of impact ionization becomes more contracted, and the ionization more predictable, resulting in a highly deterministic impact ionization event. Therefore, F(M) can be lower than 2 and McIntyre’s local field theory no longer governs such an APD. For a thick M-layer, we also have the opportunity to achieve a low k-value at a low electric field region39. Such a single carrier-initiated APD has a large difference between α and β at a low electric field, approaching a k-value of 0. Therefore, if the thickness of the M-layer is thick enough for inducing avalanche breakdown before the k-value significantly increases, we can achieve an APD with F(M) < 2. The 6 ML SL shows F(M) < 2, which is the limit of F(M) in McIntyre’s theory. It indicates that the 6 ML SL is an electron-initiated APD, and M. In other words, the F(M) of the 6 ML SL shows that multiplication process is more deterministic, and thus can be extremely low when it operates at the low electric field region where single carrier impact ionization occurs. This result is very promising, and realizing those APDs remains unfinished.From the perspective of material design, the best SL at a given 0.53Ga0.47As/Al0.48In0.52As SLs. The InGaAs/AlInAs SLs with the SL periods of 4 ML, 6 ML, and 8 ML were designed to have the same alloy contents as an Al0.4Ga0.07In0.53As RA. The electronic band structures of the SLs computed from the 14-band K.p method suggest that the k-value of the SLs is lower than that of the RA and become lower as the SL period shortens. The 4 ML SL appears to achieve the lowest k-value among them. We also computed the impact ionization rates and coefficients of the RA and the 4 ML SL. The result shows that F(M) of the SLs shows that the 8 ML SL has lower k-value (~ 0.22) than the RA, and the 6 ML SL achieved even lower k-value than the 8 ML SL, as predicted. The discussion of the design parameters for a low k-value SL suggested three design considerations, the position of 1st BZ, the effective mass, and the mini gaps. Further reduction in the k-value of the SLs can be achieved by varying M-layer thickness, but the realization of it remains for future research. This work is a theoretical modeling and experimental demonstration of engineering avalanche characteristics in InGaAs/AlInAs SLs and would also assist one to design the SLs with improved performance for various SWIR APD application.In summary, we report on the engineering of avalanche characteristics in In++-Al0.48In0.52As bottom contact layer , followed by a 100 nm n+-SL for the bottom cladding layer. Subsequently, the M-layer, 1000 nm of unintentionally doped (UID)-SL, was grown to be sandwiched by a 300 nm p+-SL top cladding layer and the bottom cladding layer.The p-i-n SLs with the periods of 6 ML and 8 ML were grown in a RIBER Compact 21DZ molecular beam epitaxy (MBE) chamber on n-type sulfur doped-InP (001) epi-ready substrates. The device structure consists of a 500 nm nk-value. Finally, a 20 nm p++-In0.53Ga0.47As top contact layer was deposited on top of the device structure to ensure an ohmic contact. A detailed diagram of the epitaxial device structure appears in Fig. It should be noted that the thickness of the top cladding layer is chosen to be thicker than that of the bottom one to ensure a single carrier (electron) injection for proper measurement of the 3PO4 (10 mL)/H2O2 (10 mL)/H2O (240 mL), was used to form the mesa. Step 3 repeated step 1 to form the bottom contact. In step 4, the SL sample was treated with HCl solution to remove the oxide layer on the mesa sidewalls before SU-8 passivation.Single-pixel devices, as shown in Fig. th and 1st order satellite peaks and are almost perfectly matched to the designed values described in Table Prior to the growth of p-i-n SLs, the thickness and composition of each SL constituent ternary alloy were accurately calibrated using reflection high electron energy diffraction (RHEED) and x-ray diffraction (XRD), respectively. Figure F(M) measurement, the devices were biased with a Keithley 2400 source meter. A 543 nm He–Ne laser was used to illuminate the APDs to ensure optical absorption near the device surface and provide near-pure electron injection profile for the 6 ML and 8 ML SLs. A standard RF Bias-Tee was used to isolate different signals in the system: the AC component of the output current was measured with an Agilent 8973A noise figure analyzer while still supplying a DC bias to the devices. The noise figure analyzer was calibrated via an Agilent 346A noise source to remove background noise.For the


Escherichia coli O157:H7 produces toxins causing serious diseases. However, traditional methods failed in detecting E. coli O157:H7 cells in the viable but non-culturable (VBNC) state, which poses a threat to food safety. This study aimed at investigating the formation, control, and detection of the VBNC state of E. coli O157:H7. Three factors including medium, salt, and acid concentrations were selected as a single variation. Orthogonal experiments were designed with three factors and four levels, and 16 experimental schemes were used. The formation of the VBNC state was examined by agar plate counting and LIVE/DEAD® BacLightTM bacterial viability kit with fluorescence microscopy. According to the effects of environmental conditions on the formation of the VBNC state of E. coli O157:H7, the inhibition on VBNC state formation was investigated. In addition, E. coli in the VBNC state in food samples  was detected by propidium monoazide–polymerase chain reaction (PMA-PCR) assays. Acetic acid concentration showed the most impact on VBNC formation of E. coli O157:H7, followed by medium and salt concentration. The addition of 1.0% acetic acid could directly kill E. coli O157:H7 and eliminate its VBNC formation. In crystal cake, 25, 50, or 100% medium with 1.0% acetic acid could inhibit VBNC state formation and kill E. coli O157:H7 within 3 days. The VBNC cell number was reduced by adding 1.0% acetic acid. PMA-PCR assay could be used to detect E. coli VBNC cells in crystal cake with detection limit at 104 CFU/ml. The understanding on the inducing and inhibitory conditions for the VBNC state of E. coli O157:H7 in a typical food system, as well as the development of an efficient VBNC cell detection method might aid in the control of VBNC E. coli O157:H7 cells in the food industry.As a common foodborne pathogen,  It was streaked on LB agar plate and grown at 37°C for 24 h. Then, a single colony was inoculated into 2 ml of LB broth and incubated at 37°C for 12 h using a shaker incubator set at 150 r/min. The bacteria suspension was diluted 1:100 in fresh medium and cultured for 4 h to the log phase upon further experiments.E. coli O157:H7, and the principle of reverse adjustment, conditions that are unfavorable to bacterial growth were selected as candidate factors to induce the VBNC state. Three factors including nutritional state, salt concentration, and acidity were taken as a single variable. To investigate possible VBNC induction conditions, three factors and four levels of orthogonal experiments were designed, and 16 protocols  was used. Five hundred microliters of the non-culturable bacterial cell sample was centrifuged at 5000 r/min for 15 min, washed with saline twice, and resuspended in saline. Subsequently, 1.5 μl of SYTO 9 dye and 1.5 μl of propidium iodide were added to the sample, followed by mixing and 30 min incubation in the dark. After incubation, 5-μl cells were captured between a slide and a coverslip and used for fluorescence microscopy. The appearance of green cells indicates the existence of VBNC cells.To determine if the non-culturable cells are in the VBNC state after exposure to the respective treatment conditions, the LIVE/DEADE. coli O157:H7 was adjusted to 107 CFU/ml. The bacterial solution with a volume of 30 ml was stored at 4 and −20°C, respectively. The number of cultivable bacteria was measured by plate counting every 3 days  in China, 25 g of crystal cake was added to 225 ml of saline, and the concentration of this medium was determined to be 100, 25, and 50% of the food sample culture medium and is configured by dilution and used after autoclaving. Subsequently, E. coli in the VBNC state was added. Bacterial suspensions with initial concentrations of 106, 105, 104, 103, 102, and 10 CFU/ml were obtained. Five hundred microliters of the bacteria suspension was added to a 1.5-ml centrifuge tube, and the PMA working solution was added to a final concentration of 5 μg/ml (to distinguish between the VBNC state and dead cells). After mixing, the samples were kept at room temperature for 10 min in the dark. Subsequently, the centrifuge tube was placed on ice, and 15 cm away from a 650 W halogen lamp for 5 min to complete the binding of PMA and DNA. The PMA–DNA was centrifuged at 10,000 r/min for 5 min, and the supernatant was discarded. DNA was then extracted using a bacterial group DNA extraction kit . The extracted DNA was detected by PCR. PCR assay was performed in a 25-μl volume and with 0.6 μM primers . The thermal profile for PCR mixtures was 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 52°C for 60 s, and 72°C for 90 s and a final extension cycle at 72°C for 7 min. The amplified products (5 μl/well) were analyzed by gel electrophoresis in 2% agarose gels and stained with ethidium bromide for 10 min. A negative control was included using sterile water instead of culture or DNA template.Twenty-five grams of crystal cake was mixed with 225 ml of physiological saline and sterilized, and E. coli O157:H7 was reduced to 5 × 104 CFU/ml in 66 days. When stored at −20°C, the culturable cell number of E. coli O157:H7 dropped to 0 in 3 days  and 8 , the culturable cell number of E. coli O157:H7 reduced to 0 in 3 days. However, in induction solutions 9  and 14 , E. coli O157:H7 cells survived for more than 57 days. Especially in induction solution 9, the E. coli O157:H7 cells survived for more than 66 days. In induction solutions 4, 10, and 13, the medium concentration is 100%. In induction solution 4 , E. coli O157:H7 all died in 3 days and did not enter the VBNC state. In induction solutions 10  and 13 , E. coli O157:H7 cells were able to survive for 33–48 days. These results suggest that the effect of acetic acid concentration on the survival of E. coli O157:H7 is stronger than that of salt concentration.In the 16 induction solutions, factors including salt, acetic acid, and medium concentrations were applied and analyzed on the effect on the survival of E. coli O157:H7 cells under a certain salt concentration were investigated. In induction solutions 1, 2, 3, and 4, the salt concentration is 0.9%. In induction solutions 1  and 2 , E. coli O157:H7 cells survived for more than 66 and 18 days at 4°C, respectively. However, in induction solutions 3  and 4 , the E. coli O157:H7 cells all died in 3 days. It showed the effect of acid on the survival of E. coli O157:H7 is stronger than that of nutrients. In addition, when the acid concentration is 1% in induction solutions 4, 8, 12, and 16, E. coli O157:H7 cells died in 3 days regardless of the medium and salt concentrations. Especially in induction solution 4  with the premium centration of medium and acid, E. coli O157:H7 cells still died in 3 days, indicating that acidity has the most significant effect on the survival of E. coli O157:H7 cells.Second, the effect of acid and medium conditions on the survival of E. coli O157:H7 were investigated. In induction solutions 6, 10, and 14, the acetic acid concentration is 0.3%. In induction solution 6 , E. coli O157:H7 cells died within 3 days. However, in induction solutions 10  and 14 , although the salt concentration increased, E. coli O157:H7 cells were able to survive from 33 to 66 days due to the high medium concentration. It indicated that the increase in the medium concentration eliminated the adverse effect of the increase in salt concentration. Among the factors on the survival of E. coli O157:H7, the effect of the medium concentration might be greater than that of the salt concentration.Third, under the same acetic acid concentration, the effects of salt and medium concentrations on the survival of E. coli. The major external protein omp  of E. coli is regulated by envZ (osmotic pressure sensor protein) and ompR. Studies have showed that wild-type, ompF, and ompC mutant strain of E. coli can enter the VBNC state under environmental pressure , but in the envZ mutant strain, they cannot enter the VBNC state. This shows that the envZ mutant strains cannot sense changes in the external environment. When the strains are exposed to adverse conditions, they cannot enter the VBNC state (relA and spoT are identified to upregulate in the VBNC state of E. coli O157:H7 (RelA or SpoT increased. (p) ppGpp synthesis increased and degradation decreased. In addition, exopolyphosphatase (PPX) was degraded by (p) ppGpp of polyphosphates , 4 , and 6 . When the acetic acid concentration is 0.7%, E. coli O157:H7 could survive in induction solutions 1  and 3 . In induction solution 5 , E. coli O157:H7 cells died within 3 days. When the medium concentration is higher than 50%, treatment with 1.0% acetic acid directly killed E. coli O157:H7 without entering the VBNC state , 4 , and 6 . When the medium concentration is 0, in induction solutions 1  and 5 , the number of culturable and viable cells was both 0 in 3 days. In induction solution 3 , E. coli O157:H7 cells were able to survive for more than 3 days. Under weak acid and high salt conditions, the formation of the VBNC state of E. coli O157:H7 was controlled by changing the nutritional condition , it ensures the elimination of E. coli O157:H7 and its VBNC state and avoid potential food safety risks.The foodborne microorganisms in the VBNC state have food spoilage ability. Compared with normal state, VBNC state cells have reduced metabolic activity and increased resistance of the cell wall to external environmental stress, including high concentrations of antibiotics, heavy metal ions, high temperature, high salt, and higher acidity . Due to E. coli in food system. Propidium monoazide (PMA) is a photoreactive DNA-binding dye. Dead microorganisms lose their capability to keep their membranes intact, which leaves the “naked” DNA in the cytosol ready to react with PMA. DNA of living organisms are not exposed to the PMA, as they have a complete cell membrane. DNA extraction and PCR amplification of PMA-treated samples can effectively detect bacteria in the VBNC state. The detection limit was determined by detecting different concentrations of E. coli O157:H7 in the VBNC state. In this study, the detection limit of the VBNC state E. coli O157 in the crystal cake food system using PMA–polymerase chain reaction (PMA-PCR) was 104 CFU/ml, which can effectively avoid the potential threat brought by VBNC state bacteria.The risk of food safety issues can be partially reduced by the efficient detection of the VBNC state of E. coli O157:H7 was as follows: acidity > nutritional state > salt concentration. When the nutrient concentration is higher than 50%, E. coli O157:H7 was killed by adding 1.0% acetic acid. In the crystal cake system, when the nutrient concentration is 25, 50, and 100%, E. coli O157:H7 cells died in 3 days by adding 1.0% acetic acid with no VBNC cells identified. In the food system, the VBNC state formation of E. coli O157:H7 was inhibited by adding 1.0% acetic acid. In addition, PMA-PCR assays can be utilized in the detection of the VBNC state of E. coli in the food system.The effect of external environmental conditions on the formation of the VBNC state of All datasets presented in this study are included in the article/supplementary material.JL and KW conceived the study and participated in its design and coordination. YML and T-YH performed the experimental work. CY and LC analyzed the data. YL and JL prepared and revised this manuscript. All authors reviewed and approved the final manuscript.The author YL was employed by Guangdong Zhongqing Font Biochemical Science and Technology Co. Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The imbalance of prenatal micronutrients may perturb one-carbon (C1) metabolism and increase the risk for neuropsychiatric disorders. Prenatal excessive methionine (MET) produces in mice behavioral phenotypes reminiscent of human schizophrenia. Whether in-utero programming or early life caregiving mediate these effects is, however, unknown. Here, we show that the behavioral deficits of MET are independent of the early life mother-infant interaction. We also show that MET produces in early life profound changes in the brain C1 pathway components as well as glutamate transmission, mitochondrial function, and lipid metabolism. Bioinformatics analysis integrating metabolomics and transcriptomic data reveal dysregulations of glutamate transmission and lipid metabolism, and identify perturbed pathways of methylation and redox reactions. Our transcriptomics Linkage analysis of MET mice and schizophrenia subjects reveals master genes involved in inflammation and myelination. Finally, we identify potential metabolites as early biomarkers for neurodevelopmental defects and suggest therapeutic targets for schizophrenia. Chen, Alhassen et al. show that schizophrenia-like behavioral deficits induced by excessive prenatal methionine administration are due to in-uterus aberrations rather than through early life mother-infant interaction in mice. This study identifies the brain metabolites and transcriptomic signatures, which potentially serve as early biomarkers for schizophrenia-like behaviors. C1 metabolism encompasses two complex pathways, the transmethylation (methionine) and the folate cycles, through which a carbon unit is transferred from one to the other metabolic pathways. Dietary or supplementary methionine is converted to S-adenosyl-methionine (SAM), the main methyl donor that is used in almost all methylation reactions, where it is converted to S-adenosyl-homocysteine (SAH)  metabolism activity during pregnancy is supported by the role of micronutrients such as methionine, choline, vitamin B12, betaine, and folate in brain development through involvement in methylation processesAH) Fig. . SAH is 15.Fluctuations in the methyl-donor dietary intake during pregnancy have been shown to cause changes in the components of the C1 pathways and in the levels of DNA methylation22. We recently examined the effects that disruption of C1 metabolism during gestation have on mice progeny. Pregnant mice were administered methionine equivalent to twice their daily dietary intake in their final week of gestation at the time of brain development23. The resulting pups exhibited a constellation of behavioral, electrophysiological and transcriptomic changes consistent with schizophrenia. For example, MET mice displayed social deficits, cognitive impairments and augmented stereotypy, accompanied with decreased synaptic plasticity, and reduced local excitatory synaptic connections in the hippocampus. The schizophrenia-related genes Npas4, Arc, and Fos were downregulated in the MET mice. Perturbation of C1 metabolism micronutrients during pregnancy has important epidemiological and therapeutic implications because of the exceptionally high potential impact on prevention or therapeutic intervention of psychiatric disorders. Therefore, the aim of this study is to investigate the early changes in the brain metabolome and transcriptome linked to methionine overload during the third week of pregnancy in mice, and whether the behavioral phenotype induced by excessive prenatal methionine can be rescued by caregiving during early life stages. The timing of the MET administration in the third week of mouse pregnancy was selected to correspond to the comparable stage of human pregnancy. Given the alterations in methyl-donor requirements and methionine metabolism during the different gestation stages, a higher rate of transmethylation occurs in the 3rd trimester of gestation in pregnant women to meet the increase in methylation demands24. Further, maternal hyperhomocysteinemia (as a result of the disrupted methionine metabolism) during the third trimester of pregnancy has been be associated with a two-fold increase in the risk of schizophrenia in the adult offspring22.The imbalance of methyl-donor micronutrients during gestational and development stages may lead to perturbations of C1 metabolism. C1 metabolism impairments increase the risk for the development of neuropsychiatric deficit such as cognitive deficits, schizophrenia and autism spectrum disorders (ASD), and produces in animals permanent phenotypic changes that resemble those in the major neuropsychiatric disordersP > 0.05  displayed similar behavioral phenotypes to those born to- and raised by their MET biological mothers (MET → MET): the MET → SAL displayed increased locomotor activity , 20 genes were upregulated: Kcnma1, Rian, Rps8, Snord38a; Serpina3n, Akr1e1, Sdf2l1, Rpa3, Sesn2, Gfap, Gpr17, Mir540, Mir669j, Mir690, Pcsk2os1, Trbj2-7, Ighm, Nfya, Noxred1, and CK137956; and 14 genes were downregulated: Zfp125, Rpl13 (Snord68), Tmem181c-ps, Ighv1-77, Tpd52l1, Rpl7a-ps5, Tmem181b-ps, Alyref, Rps6-ps2, mt-Tf, Nhp2l1, and Erdr1. Using arbitrary fold change (FC) cutoffs of ≥5 and significance P-values of 0.01, only Kcnma1 showed a statistically significant change  in whole brain tissues , phospholipids (phosphatidylcholine), and the mitochondrial molecule Malonyl-carnitine.The failure of SAL mothers to rescue the behavioral deficits in the MET-pups raises the question of which in-utero changes in the pups’ brain metabolites are responsible for such long-lasting behavioral deficits induced by prenatal excessive methionine. The global brain metabolomics’ analysis of 24 h-old pups reveals alterations in the MET pups’ brain in the components of one-carbon metabolism including SAM, betaine, glycine, glutathione, methylglycinate, and serine. Given that exogenous methionine is converted to SAM in the pregnant mice and their fetuses, the increase in SAM in the brains of the newborn MET pups’ is not surprising. SAM is the main methyl donor in the body and is used in almost all methylation reactions. Methylation substrates can be DNA, RNA, proteins, phospholipids, carbohydrates, and neurotransmitters. Therefore, excessive prenatal methionine would be expected to cause changes in all these substrates. Transcriptomic analyses reveal an increase in the expression of several genes encoding methylation enzymes that methylate DNA, mRNA, rRNA, and tRNA. MET brains also displayed increases in a number of methylated forms of molecules including amino acids  alongside the increase in LPCs, the myelination gene Gpr17 was upregulated. GPR17 is a G-protein coupled receptor that regulates oligodendrocyte differentiation in response to LPC-induced demyelination37, and is overexpressed in brain tissues experiencing demyelination injuries40; 2) arachidonic acid derivatives (such as cysteinyl leukotrienes (LTC4 and LTD4), can directly regulate GPR1743, and therefore the dysregulation of arachidonic acid pathways found in MET brains might be implicated in the demyelination; 3) the brains of newborn MET pups exhibited an increase in the mRNA and immunoreactivity of GFAP, a marker for neuro-inflammation as well as myelination;47 and 4) finally, the MET brains displayed an overexpression of Serpina3n, an astrocyte specific protein and neuro-inflammation marker. Strikingly, Serpin3a  displays the highest fold-changes in the brains of schizophrenia patients , and two-fold increase in autism 48. Serpina3 is also overexpressed in Alzheimer’s as it enhances amyloid beta fibrillization and accelerated plaque formation50.Our metabolomic and transcriptomic results reveal profound dysregulations in lipid metabolism, including membrane and mitochondrial lipids. Lipids form over 50% of the brain’s dry weight and they play crucial roles as the main brain’s cell membrane structural components, as well as energy and signaling molecules. Membrane phospholipids are the key players of myelination, neuronal membrane geometrical shape and the physical characteristics, such as the fluidity and conductance. Therefore, the disruptions observed in the MET brains’ membrane phospholipids  would profoundly impact the functions of transmembrane receptors, ion channels, and other membrane proteins. The neurotransmission signaling is also greatly impacted by abnormal lipid metabolism. Lysophosphatidylcholines (LPCs), byproducts of the phosphatidylcholine → arachidonic-acid hydrolysis, are potent membrane-dissolving molecules, and are known to induce demyelination53. Therefore, the accumulation of malonyl-carnitine in the MET pups’ brains indicates a specific disruption of fatty-acid oxidation caused by impaired entry of long-chain acylcarnitine esters into the mitochondria. The decreases of mRNAs of Cpt1 and Mcat  in the MET pups support an impaired synthesis and entry of long-chain acylcarnitines into the mitochondria. Alongside the disruption of mitochondrial lipid oxidation, metabolomic and transcriptomic analyses revealed dysregulated mitochondrial energy production. NAD+ dependent redox reactions were dysregulated, and 21 and 8 genes that are involved in NAD+ and glutathione pathways, respectively, were dysregulated in the MET brain.The mitochondrial lipid metabolism was also impacted by prenatal methionine. The metabolite that exhibited the highest change was malonyl-carnitine (increased 17-fold in the MET brains). Malonyl-carnitine concentrations reflect the levels of malonyl-CoA, a key regulator of fatty acid oxidation, which inhibits carnitine palmitoyltransferase 1 (Cpt1) and attenuates the mitochondrial uptake of long chain fatty acids56, the activation of postsynaptic BK, which couples to GluN1, suppress NMDA-potentiated evoked excitatory postsynaptic potentials (EPSP)57. Our results suggest that early upregulation of Kcnma1 channels might provoke sustained hypoactivity of excitatory pathways in the hippocampus . In agreement, Arc expression was decreased in the brains of MET newborn pups. Interestingly, the MET pups exhibited an increase in the levels of glutamate-derived neurotransmitter, N-Acetylaspartylglutamate (NAAG), which acts as an agonist at glutamate metabotropic receptors 3 (mGluR3)59 and has been associated with the pathogenesis of schizophrenia64.MET pups displayed disruptions in the amino acid neurotransmitters’ pathways and neurotransmitters’ precursors, including decreased glutamine, glutamate, tryptophan, tyrosine, and increased aspartate. The decrease of glutamate was associated with the dysregulations of 17 genes involved in glutamate neurotransmission including glutamate transport, synthesis, release, receptors, and the regulation of NMDA receptors (Kcnma1). Kcnma1 displayed the highest increase in gene expression in MET brains (7-fold), and immunohistochemistry results revealed higher number of Kcnma1-expressing neurons in the hippocampus, thalamus and amygdala of MET brains. Kcnma1 encodes the large-conductance calcium-activated potassium channel, subfamily M, alpha member 1 (BK channel) pore-forming alpha subunit, which is involved in neuronal excitability and plasticity, and has been associated with neurological and psychiatric disorders, including epilepsy autism and Alzheimer’s. While the activation of presynaptic BK channels decreases glutamate releasep values of the corresponding Fisher tests are close to 1).While our metabolomics and transcriptomic results support the phospholipid, mitochondria and glutamate dysfunction hypotheses of schizophrenia, these results point at similar changes occurring in schizophrenia and autism  as evidenced by the linkage analysis of the transcriptomics of the MET mice with the gene expression changes in schizophrenia and autism patients. The results, thus, suggest that the two neurodevelopmental disorders may partially share some etiological origins. The linkage results, however, should be cautiously interpreted, as the analysis of the intersections’ size showed no significant difference from what can be expected by chance  at a dose of 750 mg/kg (Sigma-Aldrich) or saline (SAL) twice a day for 7–8 consecutive days from gestational day 14 until delivery. We chose a methionine dose and treatment schedule that were used previously by other groups68, and then by our group25, which corresponded to the regimens previously administered to humans, and was reported to exacerbate the psychotic symptoms in schizophrenia patients70.Swiss Webster mice, 8–9 weeks’ age, were obtained from Charles River Laboratories . One male and two female mice were group-housed, and pregnant mice were individually housed from the thirteen day of pregnancy  until delivery. Pregnant mice that mated with the same male mouse were subcutaneously administered with All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of California, Irvine and were performed in compliance with national and institutional guidelines for the care and use of laboratory animals. Pups of SAL and MET mice were raised by their biological mother (SAL/SAL and Met/MET) or were cross-fostered to a female of the other treatment (SAL/MET and MET/SAL for SAL pups with MET mother and MET pups with SAL mothers respectively) within 24 h of birth. Pups remained with their biological or foster mother until they were 21 days old, then male mice were selected for the study and group-housed in groups of 2–5 mice.Maternal behavior: Nest building. Nesting building behavior was assessed on PPD1 using the 0–5 scale of nest quality72.71.The retrieval behavior test was performed at PPD2 to measure the mother’s latency to retrieve the first pup and the total time required to retrieve 3 pups as described previously23.Male mice were tested from postnatal week 8 to week 12 with a battery of behavioral paradigms in the following order: locomotion, spontaneous T-maze alternation, and prepulse inhibition. The sequence of specific assays spaced by 3–6 days inter-assay interval was adapted from our report25. Mice were allowed to habituate for 30 min in a locomotion test chamber , and locomotor activity was recorded for 1 h and analyzed by Activity Monitor 5 software .Locomotor activity was measured as described previously23. Mice were placed in the start area of the T-maze  and allowed 30 s of acclimation before the start of each trial. The door was then opened and mice were allowed to make a free choice into either side arm and to explore the arm for 30 s before being returned to the start area for the next trial. A total of eight trials were completed. Percentage of Alternation was calculated, and time to make a choice was recorded.The spontaneous T-maze alternation assay was carried out as described previously73. Mice were placed in the startle chambers for 5 min’ acclimation with 65 dB background noise. The PPI session consisted of five different trials: no-stimulus trials, three prepulse trials and startle trials. No-stimulus trials consist of background noise only (65 dB). Startle trials consist of a 40 ms duration startle stimulus at 120 dB (p120). Prepulse trials consist of a 20 ms duration prepulse at 68 dB (pp3), 71 dB (pp6), or 77 dB (pp12), a 100 msinterstimulus interval, followed by a 40 ms duration startle stimulus at 120 dB. Test sessions began with 5 presentations of the p120 trial, followed by 10 presentations of the no-stimulus trial, p120 trials, pp3, pp6, and pp12 prepulse trials. The amount of PPI is calculated as a percentage score for each acoustic prepulse intensity.The Prepulse inhibition (PPI) assay was measured as previously described74. A differential analysis of the brain metabolites was performed using the Cyber-T program between the MET-treated and control groups for both newborn and adult mice groups, to identify the top up- or down-regulated metabolites76.Metabolomics profiling on whole homogenate brain tissues collected from 24 h or 13 weeks’ old mice was carried out at the West Coast Metabolomics center . The Metabolomics profiles consisted of three platforms: (1) primary metabolites including carbohydrates and sugar phosphates, amino acids, hydroxyl acids, purines, pyrimidines, aromatics; (2) complex lipids; and (3) biogenic amines and methylated and acetylated amines. For the 24-h-old pups, tissues of one brain hemisphere  were homogenized and extracted following the protocols first published in77. Total RNA was extracted (Qiagen) from one brain hemisphere of the 24-h-old pups  according to the manufacturer’s protocol, . RNA was then reverse-transcribed into cDNA and analyzed by “whole-transcript transcriptomics” using the GeneAtlas microarray system (Affymetrix) and the manufacturer’s protocols. The newly synthesized sscDNA was hybridized using MoGene 1.1 ST array strips (Affymetrix). Each array comprised 770,317 distinct 25-mer probes to probe an estimated 28,853 transcripts, with a median 27 probes per gene. Gene expression changes associated with the methionine treatment were analyzed with Transcriptome Analysis Console (TAC) software (Affymetrix) using Tukey’s Bi-weight Average algorithm and default settings, generating fold-change and unpaired ANOVA values.Microarray experiments and analysis were performed as previously describedp < 0.05) undergo further analyses including: (1) pathway enrichment analysis using the Pathway Common Database78 and the ConsensusPathDB;79 (2) identification of transcription factors (TFs) and RNA-binding proteins as important regulators; (3) transcription factor binding site (TFBS) enrichment analysis of all promoters using the MotifMap81 database and the CHiPSeq database from ENCODE;82 (4) analyses of all downstream targets of differentially expressed TFs and RBPs81.Top up- or down-regulated genes 48. Common genes that are differentially expressed in both mouse and human autism, and both mouse and human schizophrenia are computed for adult and newborn mouse groups.The differentially expressed genes in both adult and newborn mouse groups from the transcriptomic analysis are compared with differentially expressed genes in human autism and schizophreniaTwenty-four hours’ pups were perfused transcardially with 0.9% saline and 4% paraformaldehyde. Brains were collected, post-fixed in 4% PFA for 24 h, and transferred to 30% sucrose at 4 °C. Fifty-micron sections were cut using a cryostat (Microm HM505E) and stored in phosphate buffered saline with 1% Sodium azide until immunostaining was performed. Three brain sections from four brains in each group were selected from regions at the following levels: hippocampus, amygdala, and somatosensory cortex, and thalamus. Sections were blocked with 4% goat serum in phosphate buffered saline (PBS) with 0.3% Triton x-100 for 60 min, and then incubated with the primary antibodies: rabbit anti Arc , GFAP , rabbit anti KCNMA1  at room temperature for 24 h. Sections were washed and stained with goat anti rabbit AlexaFluor488  goat anti rabbit AlexaFluor555 (1:500 Invitrogen), and DAPI  to stain the nuclei. Sections were then mounted on slides with antifade solution. Leica Sp8 confocal microscope  was used for image acquisition and ImageJ and Volocity were used for analysis.Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Reporting SummaryPeer Review File