N-methylformamide with water was measured at four temperatures, 293.15 K, 298.15 K, 303.15 K, and 308.15 K, and the standard partial molar heat capacity of cyclic ethers has been discussed. 18-crown-6 (18C6) molecules can form complexes with NMF molecules through the hydrogen bonds between –CH3 group of NMF and the oxygen atoms of 18C6. Using the model of preferential solvation, the cyclic ethers were observed to be preferentially solvated by NMF molecules. It has been proved that the molar fraction of NMF in the solvation shell of cyclic ethers is higher than that in the mixed solvent. The exothermic, enthalpic effect of preferential solvation of cyclic ethers increases with increasing ring size and temperature. The increase in the negative effect of the structural properties of the mixed solvent with increase in the ring size in the process of preferential solvation of the cyclic ethers indicates an increasing disturbance of the mixed solvent structure, which is reflected in the influence of the energetic properties of the mixed solvent.The aim of the work was to analyze the preferential solvation process, and determine the composition of the solvation shell of cyclic ethers using the calorimetric method. The heat of solution of 1,4-dioxane, 12-crown-4, 15-crown-5 and 18-crown-6 ethers in the mixture of  Scientists dealing with organic synthesis are looking for solvents in which the chemical reactions that are carried out will have the highest possible efficiency. Not only are pure solvents taken into account, but also mixed water–organic and organic–organic mixtures that significantly affect the solvation of the reactants. The solvation process plays a very important role in the interactions between molecules of solvent, solvent and solute, as well as solute and solute ,2,3. StuN-methylformamide–water (NMF + W) mixed solvent.Studies on preferential solvation have been carried out for a long time, mostly using solubility measurements ,7,8. MarCrown ethers are an interesting class of compounds, characterised by a hydrophobic–hydrophilic character. They can form selective complexes with cations and small organic compounds ,21. For N-methylformamide is a solvent with hydrophilic properties and can be treated as a derivative of formamide (F), obtained by replacing one of the hydrogen atoms connected to the nitrogen atom of formamide with a –CH3 group. The obtained results of the preferential solvation study in the NMF + W mixture can be compared with the analogous ones referring to the F + W mixture.xw in the NMF + W mixture.As can be seen in xW in the mixture. In the case of 1,4-dioxane, the values of the function xW ≈ 0.5 and then decrease. This is probably related to the structure of the molecules of cyclic ethers. The 12C4, 15C5, and 18C6 are included in crown ethers with a hydrophilic character inside the molecule and a hydrophobic character outside the molecules. The molecules of 1,4-dioxane are smaller than other cyclic ethers, and it is not possible to unambiguously locate the hydrophobic and hydrophilic nature of this part of the molecule.The course of curves is monotonic and decreases with an increase of The data of the standard molar enthalpy of solution of the investigated cyclic ethers allow for us to calculate the standard molar heat capacity of the solution of cyclic ethers using Equation (1).Then, the standard partial molar heat capacity . In this range, the increase in the function xW < 0.85), the values of the function xW ≈ 0.85. This means that, in this system, there are some changes in the interactions due to the dissolution of 18C6 molecules in the NMF + W mixture. The answer to this question is not as obvious. In the literature, we found no information on the interactions between 18C6 molecules and NMF, as was the case for interactions between 18C6 molecules and formamide (F). 18C6 molecules were found to form complexes with the F molecules in the solid state [xW < 0.1) [As seen in y Somsen  and Casty Somsen  regardiny Somsen  in the cid state ,38. In oW < 0.1) .N-methylforamide can form three hydrogen bonds with water, whereas this type of interaction is less important in the F + W mixtures [x, noticed that the mixture of NMF + W behaves almost like an ideal solution [In the NMF + W mixture, a change in the function s than F . The enes than F . When fos than F ,42. In ts than F , viscosis than F . In addimixtures . Furthersolution .3 group and an electron pair of oxygen atoms of the 18C6 molecule, similarly to the hydrogen bonds of acetonitrile with oxygen atoms of the 18C8 molecule [In general, the structure of the mixed solvent NMF + W is weaker than that of F + W; therefore, there is the possibility of a specific interaction between 18C6 molecules and NMF molecules in a wider range of the mixture composition than in the case of the F + W mixture. On this basis, it can be assumed that 18C6 molecules form complexes with NMF molecules through hydrogen bonds. We can suppose that 18C6 molecules form complexes by hydrogen bonds between hydrogen atoms of the NMF molecule and an electron pair of oxygen atoms of the 18C6 molecule, as well as between hydrogen atoms of the –CHmolecule , particuThe cyclic ethers, because of their hydrophobic character, are hydrophobically hydrated in mixed solvent with a high water content. In addition, the molecules may be preferentially solvated by the organic component of the mixture.In order to calculate the enthalpy effect of preferential solvation, we used the model proposed by Covington ,18 and mHb(W) − the enthalpic effect of hydrophobic hydration of the solute in pure waterBy rearranging Equation (3), we can calculate the enthalpic effect other than hydrophobic hydration: n and Hb(W), which are necessary for the calculations, were taken from our publication [The method of calculating the enthalpic effect of hydrophobic hydration was described in our previous publication . The vallication .In the case of investigated cyclic ethers within the whole mixed-solvent composition range, the value of function negative . TherefoAs shown in i ≤ r).For a further analysis of the obtained results, we used the theory of preferential solvation ,18, hopiKi described the process of changes in the structure of the solute solvation sphere (S) caused by the changes in the mixed solvent composition because of preferential solvation. This can be represented by Equation (6).K—the overall constant of equilibrium of all processes, The equilibrium constant The enthalpic effect of the preferential solvation of the process presented by Equations (5) and (6) is described by Equation (7).r and Kr1/ were calculated and are presented in Using Equation (7) and non-linear regression, parameters Kr1/ is shown to be lower than 1, which means that the functional groups in cyclic ethers are preferentially solvated by the organic component (NMF) of the mixture [r increase with increases in the ring size of cyclic ethers and decrease with increasing temperature.In  mixture . MoreoveK, described by Equation (5), represents the reverse process, i.e., the solvation by water molecules. Thus, in the case of preferential solvation by NMF molecules, the equilibrium constant of this process will be equal to K’ = 1/K  and oxygen atoms in the cyclic ether molecules, especially in the areas of medium- and low-water content in the mixture. In addition, molecules of 18C6 can form complexes with NMF molecules by interacting with the –CHThe thermodynamic function  of transferring substance (S) from W to the mixed solvent NMF + W in the preferential solvation process can be described by Equations (8)–(10), proposed by Balk and Somsen .(8)ΔtrGrW, were calculated using Equation (10) by choosing the rW values so that equality was met: N-methylformamide in the solvation sphere, rNMF, and the mole fraction of NMF, yNMF, in the solvation sphere were calculated. yNMF, is almost independent of temperature, so the mean value was calculated for each cyclic ethers and is presented in xW, and NMF, xNMF in the NMF + W mixture are also presented. Using Equation (11) the values of N-methylformamide in the solvation sphere (yNMF) are higher than those in the mole fraction of N-methylformamide in the NMF + W (xNMF) mixture. Thus, it is clear that cyclic ethers are preferentially solvated by N-methylformamide molecules.In For 1,4-dioxane, the values of the mole fraction of NMF in the solvation sphere are higher than those in the mole fraction of F in this sphere ; howeverIn our previous paper , we propbVE and cHE, have been calculated and are presented in The excess molar volume  with water is observed in the case of the enthalpic effect, which differs to the hydrophobic hydration of 15C5 . HMPA isIn our case, NMF interacts with water through hydrogen bonds and forms a new, stable structure with water, which is also reflected by the negative contribution of the structural properties and the positive contribution of the energetic properties of the mixed solvent to the solvation of the cyclic ethers.N-methylformamide) are shown in Suppliers, purity, a method of purification and the water content of the compounds used for measurements  at four temperatures led to the following conclusion regarding the solvation of the cyclic ethers.The investigation of the dissolution enthalpy of cyclic ethers  in the mixture of The exothermic enthalpic effect of the solvation process increases with the increase in the size of the cyclic ethers ring.N-methylformamide and water.Molecules of 1,4-dioxane, 12C4, 15C5, and 18C6 are preferentially solvated by the NMF molecules in a mixture of The exothermic, enthalpic effect of preferential solvation increases with increasing temperature for the cyclic ethers .The total number of NMF and W molecules in the solvation sphere of the molecules of cyclic ethers increases with increasing ether ring size.The mole fraction of NMF in the solvation sphere of cyclic ethers is higher than that of the molecules in the NMF + W mixture.The increase in the negative effect of the mixed solvent’s structural properties with the increase in the size of the cyclic ring during the process of preferential solvation of the cyclic ethers indicates an increasing disturbance to the mixed solvent structure, which is reflected in the influence of the energetic properties of the mixed solvent.


A three-dimensional transient numerical simulation of the flow boiling of ADN-based liquid propellant in the capillary tube was carried out using the VOF (Volume of Fluid) coupled Lee model. The flow-solid temperature and the gas–liquid two-phase distribution and the wall heat flux at different heat reflux temperatures were analyzed. The results show that the magnitude of the mass transfer coefficient of the Lee model significantly influences the gas–liquid distribution in the capillary tube. The total bubble volume increased from 0 mm3 to 957.4 mm3 when the heat reflux temperature was increased from 400 K to 800 K. The bubble formation position moves upwards along the inner wall surface of the capillary tube. Increasing the heat reflux temperature intensifies the boiling phenomenon. When the outlet temperature exceeded 700 K, the transient liquid mass flow rate in the capillary tube was already reduced by more than 50%. The results of the study can be used as a reference for the design of ADN-based thruster.During the operation of ADN (ammonium dinitramide, (NH In recent years, with the development of the economy and the increasing demand for low carbon and environmental protection, the production of high energy and green energetic materials has become a hot research issue . Scienti4+) and the dinitramide anion (N(NO2)2−) . (NO2)2−) . The decuid flow .Previous studies have shown that the effect of surface tension gradually increases with decreasing channel size, and that micro-scale flow boiling can exhibit a completely different pattern to that of conventional channels. Within the micro-scale channel, the effect of local breakage and coalescence of the gas–liquid interfaces on mass and heat transfer can become significant . In geneIn the study of flow boiling in microchannels, A. Mukherjee  analyzedThe objective of this paper is to investigate and elucidate the effect of heat reflux temperature (400 K~800 K) on the boiling heat transfer of ADN-based liquid propellant flow in the capillary tube through computational fluid dynamics (CFD) simulations. The ANSYS Fluent 19.2 CFD software was used for the numerical simulation to calculate the flow boiling process of ADN-based liquid propellants using the VOF coupled Lee model. In The ADN-based thruster consists mainly of a valve seat, flange, support ring, capillary tube, front chamber, thrust chamber, and nozzle, the geometric model of which is shown schematically in The dimensional parameters of the geometric model of the capillary tube are given in Before running the simulation, the grid independence is first verified, and the effect of the grid number on the simulation accuracy is shown in In this study, the following assumptions were made on the premise that the boiling process of the ADN-based liquid propellant flow in the capillary tube can be accurately described: (1) the flow state of the ADN-based liquid propellant in the capillary tube was determined to be turbulent flow; (2) the ADN-based liquid propellant gas–liquid two-phase flow was determined to be an incompressible flow; (3) the surface tension between the gas and liquid phases was considered.−5~10−6 s and a global Courant number  model proposed by Brackbill et al. . Its expWhen heat from the combustion chamber is transferred through the wall to the upstream capillary tube region, there are physical phenomena such as heat conduction within the solid part of the capillary tube; fluid–solid coupling heat transfer and heat radiation from the solid to the environment occur. This heat transfer process is the basis for numerical simulations to obtain accurate boiling results for the flow in the capillary tube.T is the temperature of the solid in K; The energy equation for heat transfer within the solid is shown as follows :(14)ρscsAt the fluid–solid coupling interface, the fluid–solid heat flow conservation should be satisfied :(15)KconThe P1 radiation model  is used n is the refractive index of the medium; The transmission equation is shown below:The final expression of the P1 radiation model is obtained by combining the above equations.The static contact angle for wall adhesion calculation in ANSYS Fluent was determined using the sessile drop, the most commonly used contact angle measurement method . The ADNIn order to verify the accuracy of the established numerical model, numerical simulations of the flow boiling of ADN-based liquid propellant in the capillary tube were performed at a heat reflux temperature of 800 K, and the capillary inlet and outlet pressure drop of 0.5 MPa. The simulated inlet mass flow rate and the temperature at the outer wall surface of the capillary tube (at about 4.3 mm from the capillary tube outlet) were compared with the experimental results. The same capillary structure and boundary conditions were used for the simulation and the experiment. The experimental system  consistsThe relative error of the temperature of the outer wall surface at a distance of approximately 4.3 mm from the capillary tube outlet is:The relative error of the capillary tube inlet mass flow rate is:7 s−1, and the value of the mass transfer coefficient is related to the grid size, time step, specific phase transition phenomena, and other factors [In this study, the Lee model is used to simulate the flow boiling process of ADN-based liquid propellant in the capillary tube, and the specific expressions of the Lee model can be seen in Equations (2)–(10). The Lee model involves the problem of taking the value of the mass transfer coefficient. In general, the value of the mass transfer coefficient ranges from 0.1 to 10 factors . The mas factors ,29,30, w factors ,32,33. T3; whereas, when the mass transfer coefficient was 100, the total bubble volume generated reached about 953 mm3.−1, which means that the accuracy of the simulation can be well maintained, and a better convergence can be ensured.Combined with the literature, it is known that for the flow boiling problem, Z. Yang et al.  proposedIn this study, the phase change heat transfer process of ADN-based liquid propellant in the capillary tube is investigated at different heat reflux temperatures, and five numerical simulation conditions are shown in 6 W/m2. When the heat reflux temperature is 400 K, the maximum amplitude of the wall heat flux is 3 × 106 W/m2. The amplitude is reduced by about 57%. As the heat reflux temperature gradually increases, more bubbles are generated by the phase transition of ADN-based liquid propellants in the capillary tube. As a result, the amplitude of the wall heat flux in the capillary tube gradually increases as the bubbles form and coalesce. Additionally, with the gradual increase in frequency, the amplitude of the wall heat flux density shows a tendency to gradually decrease.3 at 500 K to 1.83 mm3 at 800 K.3, 50 mm3, 286 mm3, 505 mm3, 702 mm3), and the increase in the heat reflux temperature intensifies the phase change in the ADN-based liquid propellant in the capillary tube, so that more bubbles are generated in the unit time.Combined with the bubble formation position curve analysis, it was found that with the gradual increase in the heat reflux temperature, the bubble formation area in the capillary tube gradually moved upstream. When the thermal immersion temperature is 400 K, the saturation temperature of ADN-based liquid propellant in the capillary tube flow state has not been reached; therefore no phase change at this temperature, the flow state for the ADN-based liquid propellant based single-phase flow; when the heat reflux temperature exceeds 500 K, there will be a phase change, bubble formation zone from the capillary tube outlet gradually moved upstream. The bubble formation zone moves from 0.6 mm at 500 K to 2.0 mm at 800 K (distance from the capillary tube outlet length).In order to clarify the effect of the heat reflux temperature on the gas–liquid two-phase flow distribution in the capillary tube, the characteristics of the gas–liquid phase distribution in the capillary tube at 200 ms were analyzed, as shown in In this study, the effect of mass transfer coefficient in Lee model on ADN-based liquid propellant flow boiling is analyzed by numerical simulation for the first time, and the mass transfer coefficient applicable to ADN-based liquid propellant flow boiling is determined. The heat reflux immersion temperature is revealed for the gas–liquid two-phase flow and the temperature distribution law in capillary tube, which has some guidance for the design of ADN-based thrusters. The main conclusions can be drawn as follows.(1)Simulation of the flow boiling of ADN-based liquid propellant in the capillary tube can be achieved by using the VOF-coupled Lee model. The inlet mass flow rate is about 0.0300 g/s, with an error of about 5.3% from the experimental result (0.0308 g/s), and the outer surface temperature at about 4.3 mm from the capillary tube outlet is about 420.1 K, with an error of about 2.6% from the experimental result (398.7 K), which meets the accuracy requirement;(2)3 to 953.0 mm3), and the gas–liquid equivalence surface gradually becomes complete. For the flow boiling problem of ADN-based liquid propellant in the capillary tube, the mass transfer coefficient is taken as 100 s−1 to maintain the accuracy of the simulation well and to ensure a good convergence;The mass transfer coefficient in the Lee model affects the frequency of bubble generation in the capillary tube under the same simulation conditions. As the mass transfer coefficient gradually increases (from 0.1 to 100), more volume bubbles are generated in the capillary tube (from 4.0 mm(3)During the transfer of the heat reflux temperature upstream, the temperature of the front chamber and the outer wall surface of the capillary tube are uniformly distributed, and the temperature is symmetrically distributed along the axial direction. Due to the cooling effect of the ADN-based liquid propellant, the temperature of the inner wall surface of the capillary tube is slightly lower than that of the outer surface;(4)3 to 702 mm3). The time of the first bubble formation is gradually advanced, and the bubble formation position moves upstream;The increase in the heat reflux temperature (from 400 K to 800 K) intensified the flow boiling in the capillary tube, and the average bubble volumes in the capillary tube increased significantly (from 0 mm(5)As the heat reflux temperature increases, the local boiling phenomenon of the ADN-based liquid propellant in the capillary gradually intensifies. When the outlet temperature exceeded 700 K, the transient liquid mass flow rate in the capillary was already reduced by more than 50%. In the design of the thruster structure, technologies such as adding insulating layers or thermal coatings to the capillary outlet are used to ensure that the temperature at the capillary outlet area does not exceed 700 K;(6)In this study, the ADN-based liquid propellant physical parameters are assumed to remain constant. In fact, the physical parameters change with temperature. The physical parameters  of ADN-based liquid propellants at different temperatures will be measured experimentally in later studies to verify the model in this paper in detail.


This study aimed to evaluate whether CT angiography (CTA) manifestations in anterior cerebral artery a1 segment (A1) were related to the hemodynamics in patients with internal carotid artery stenosis (ICAS).A total of 97 cases were selected. The degree of ICAS and symmetry of A1 were evaluated by CTA examination. Hemodynamic indexes were detected by transcranial Doppler (TCD). The differences in CTA presentations of A1 and hemodynamics between the vessels on the stenotic and contralateral sides were analyzed according to the different degrees of stenosis. The degree of ICAS according to the different manifestations of A1 and the hemodynamics of A1’s adjacent vessels were also analyzed.P < 0.001).In the case of unilateral ICAS, the difference in Vm of A1 between the stenotic and the contralateral side was the most significant relative to the stenosis degree. When unilateral ICAS was ≥70%, the presentation of A1 on the stenotic side was more slender or non-visualized compared to that on the contralateral side, while in cases with unilateral stenosis <70% or bilateral stenosis with a similar degree of stenosis, A1 were mainly symmetrical. When A1 on the side of ICAS was slender or non-visualized, the Vm of A1 was significantly slower than that on the contralateral side (The CTA manifestations of A1 on the side of ICAS embodied the overall changes of the intracranial hemodynamics after ICAS. A combination of TCD and CTA examination of A1 can assist in judging the location and degree of ICAS. In our clinical practice, asymmetry of the anterior cerebral artery a1 segment (A1) bilaterally was commonly found in CT angiography (CTA) examination in patients with internal carotid artery stenosis (ICAS). The circle of Willis is an important channel for the compensation of intracranial blood flow in ICAS . PreviouCases were selected from the Department of Neurosurgery, Shengjing Hospital of China Medical University. The degree of ICAS and the imaging manifestations of main intracranial and extracranial vessels were determined by CTA examination. The stenosis site of internal carotid artery (ICA) was located at the bifurcation of common carotid artery (CCA) in enrolled cases. The degree of ICAS was calculated according to the All enrolled patients underwent TCD for the detection of major intracranial and extracranial vessels that included the CCA, ICA, middle cerebral artery (MCA), A1, posterior cerebral artery (PCA), ophthalmic artery (OA), supratrochlear artery (StrA) and siphonage of internal carotid artery (CS). A digital dual-channel, multi-depth TCD instrument and pulse Doppler probe  were used. Bilateral MCAs, A1 branches and PCAs were detected in the supine temporal window, and bilateral OAs and CSs were detected in the orbital window . Bilateral CCAs, proximal ICAs, and StrAs (at the inner canthus) were detected in the supine position with a 4.0 Hz probe. In addition, cervical compression was used to determine the opening of the ipsilateral posterior communicating artery (PcoA). The blood flow direction, systolic and end diastolic blood flow velocity were measured, and the mean blood flow velocity (Vm) and pulse index (PI) were calculated .The A1 was classified into three categories according to the imaging characteristics of bilateral A1 in CTA, namely: symmetrical A1; unilateral slender A1 ; and unilateral A1 that was not visualized on imaging.The unilateral and bilateral stenosis cases were first grouped according to the degree of ICAS that was classified according to the CTA by NASCET criteria. Unilateral stenosis cases were classified into five groups according to the degree of stenosis that were 50–69%, 70–79%, 80–89%, 90%–near complete occlusion, and complete occlusion. According to the group, the Vm, blood flow direction, PI value, wall shear stress (WSS) of the A1, and imaging manifestations of A1 on both sides were recorded. Finally, based on the imaging manifestations of A1, the proportion of the degree of ICAS under different manifestations of A1, the Vm of A1 and MCA on both sides, blood flow direction and PI value were recorded.t-test (two-tailed) statistical method was used to calculate the statistical differences in Vm and PI values between the ipsilateral stenosis vessels and the contralateral vessels. Multiple comparisons between groups were performed using the Bonferroni method. The Chi-square independence test was used to compare the differences in the case distribution of ICAS in different manifestations of A1. SPSS 26.0 software was used for statistical analysis, and statistical significance was considered with a P value < 0.05.When the patients were grouped according to the degree of ICAS and A1 performance, the age of the patients, Vm of each vessel and PI values were represented by the mean ± standard deviation. The paired sample The average age of the 97 cases enrolled was of 64 ± 7 years, with 82 males and 15 females. Of the 75 unilateral ICAS cases, 15 were classified into in the 50–69% degree of stenosis group, 10 in the 70–79% group, 22 in the 80–89% group, 19 in the 90%–near complete occlusion group, and 9 in the complete occlusion group. Of the 22 bilateral ICAS cases, 16 were considered unilateral severe stenosis and six were similar bilateral stenosis .P > 0.05). When the degree of stenosis was 70–79%, the Vm of the A1 on the stenotic side was lower than that of the contralateral side (P < 0.05). When the degree of stenosis was 80–89%, the difference observed in the Vm of A1 bilaterally was the most obvious (P < 0.001), and the direction of blood flow was toward the probe in 15% of these patients. When the degree of stenosis was 90% to near complete occlusion, the difference observed in the Vm of A1 bilaterally decreased (P < 0.05). In 31.58% of these cases, the direction of blood flow in the A1 on the stenotic side was toward the probe. When an ICA was completely occluded, there was no statistical difference observed in the Vm of A1 bilaterally (P > 0.05), and the direction of blood flow in the A1 of the stenotic side was toward the probe in 33.33% of these cases. It should be noted that when the degree of internal carotid artery stenosis reached over 80%, the PI value of the stenotic MCA decreased when compared with that of the contralateral side, and the opening of the OA and StrA secondary collateral circulation gradually increased. When the ipsilateral internal carotid artery was completely occluded, the OA on the stenotic side of all enrolled cases showed a reverse direction of blood flow. In addition, we found that as the degree of stenosis increased, the proportion of ipsilateral PcoA opened and supplied blood to the anterior circulation increased gradually , except for the difference in the hemodynamics when the CCA and ICA were compared. In 31.25% of the cases, the direction of blood flow of the A1 on the stenotic side was toward the probe. In 56.25% of the cases, the PcoA on the worse side was open and supplied to the ipsilateral anterior circulation. In addition, both the OA and StrA secondary collateral circulation showed varying degrees of opening in cases with both severe and relatively mild stenosis . In cases of the bilateral ICAS, the WSS of A1 on the side with more severe stenosis was lower than that on the other side (p < 0.05) (p > 0.05)   of A1 on < 0.05) , while i > 0.05) .In cases of unilateral ICAS, the manifestations of A1 observed on CTA bilaterally varied according to the degree of ICAS. When the degree of stenosis was 50–69%, most A1 images were symmetrical. When the degree of stenosis was 70–79%, bilateral A1 symmetry observed on imaging decreased. When the degree of stenosis was in the range of 80–89%, majority of A1 on the stenotic side presented as slender or non-visualized. When the degree of stenosis was 90% to near complete occlusion, the proportion of A1 on the stenotic side that were slender or non-visualized was 68.42%. However, when the ICA was completely occluded, the proportion of occluded A1 that presented as slender or non-visualized was 77.78% . In the P < 0.001). There were a total of 38 cases of unilateral slender A1 on the stenotic side, among which 11 had a degree of stenosis of 80–89% (28.95%), and 9 had 90% to near complete occlusion (23.68%). In 10 of these cases, the direction of blood flow in the stenotic A1 was toward the probe (P < 0.001). In addition, regardless of the direction of blood flow in the A1 on the stenotic side, there were statistically significant differences in the Vm and PI value for the MCA on both sides (P < 0.05). There were 26 cases with symmetrical A1 imaging, with the highest proportion of 10 cases in unilateral ICAS with a degree of stenosis of 50–69% (38.46%). In 21 cases, the direction of blood flow of the A1 on the stenotic side deviated away from the probe, and there was no statistically significant difference between the Vm and PI value of A1 and MCA bilaterally  had a degree of stenosis of 80–89%. In 17 of these cases, the direction of blood flow in the A1 on the stenotic side deviated from the probe , and thehe probe , while the probe . There wP < 0.01). The Vm in the A1 on the stenotic side was significantly lower than that on the contralateral side if the direction blood flow in A1 deviated from the probe (P < 0.001), regardless of whether the A1 on the stenotic side was slender or non-visualized.According to the symmetry or asymmetry  of A1 on both sides, it can be seen that the degree of unilateral ICAS was usually more than 70% when A1 on the side of ICAS is slender or non-visualized (CTA findings of A1 and overall changes of intracranial hemodynamics after ICAS.via collateral circulation (Collateral circulation plays an important role in the pathophysiological process of cerebral ischemia . It is pculation  and intrculation . An examculation . The ICAculation , which iculation . In our Our study found that the hemodynamic changes in A1 on the side of ICAS were related to the degree of stenosis, and the CTA manifestations of A1 may be caused by its hemodynamics. Although CTA examination is an effective method to evaluate ICAS, in clinical practice , cervicaIt is also important to note that the degree of stenosis in the ICA did not affect the Vm of the MCA on the stenotic side, which indicated that the hemodynamic changes caused by ICAS did not affect the distal vessels , 2015. HThe limitation of this study is that there are no dynamic intracranial vascular changes, and longer observation and related hemodynamic mechanism research should be conducted in the future.Although variations in the circle of Willis are common, changes in hemodynamics may affect the imaging of intracranial vessels in ischemic cerebrovascular disease, especially at the location of blood flow convection. The symmetry of A1 is relative to hemodynamics rather than variations in the circle of Willis. In addition, the activation of the primary and secondary collateral circulation is relative to the degree of stenosis. The “convection effect” proposed in this study provides a basis for understanding the hemodynamic changes and pathological mechanisms associated with ICAS. Our study found that CTA imaging of A1 on the stenotic side after ICAS embodied the intracranial hemodynamics at A1. The combined findings of the hemodynamics of A1 bilaterally and the CTA examination can assist in judging the location and the degree of ICAS.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by the Ethics Committee of Shengjing Hospital of China Medical University. The patients/participants provided their written informed consent to participate in this study.CL and WM: study concept and design, analysis and interpretation, and critical revision of the manuscript for important intellectual content. BX, ML, XL, and XD: acquisition of data, analysis, and interpretation. JM and SL: study concept and design, critical revision of the manuscript for important intellectual content, and study supervision. All authors also confirm accountability for the accuracy and integrity of the work.The 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The goal of this research is to present the possibility of synthesizing B4C nanoparticles from elements via vapor deposition and modifying the morphology of the obtained powders, particularly those synthesized at high temperatures. B4C nanoparticles were synthesized in the process of direct synthesis from boron and carbon powders heated at the temperature of 1650 °C for 2 h under argon and characterized by using scanning electron microscopy, transmission electron microscopy, atomic force microscopy, X-ray diffraction analysis, and dynamic light scattering measurements. The physicochemical characteristics of B4C nanoparticles were determined, including the diffusion coefficients, hydrodynamic diameter, electrophoretic mobilities, and zeta potentials. An evaluation of the obtained B4C nanoparticles was performed on several human and mouse cell lines, showing the relation between the cytotoxicity effect and the size of the synthesized nanoparticles. Assessing the suitability of the synthesized B4C for further modifications in terms of its applicability in boron neutron capture therapy was the overarching goal of this research.Boron carbide is one of the hardest materials in the world which can be synthesized by various methods. The most common one is a carbothermic or magnesiothermic reduction of B


Optimizing the chemical composition of phosphorus cast iron for the manufacture of brake shoes, which are used in the rolling stock, is based on specific tests, and the results must be within the specified limits of railway standards. This paper presents the processing of data taken from an economic agent producing brake shoes—with chemical composition and hardness values determined through the Brinell method—as well as optimizing these data by obtaining optimal variation intervals. These technological intervals are useful in the case of foundries in order to obtain superior products from a qualitative point of view. Brake shoes for rolling stock are made of P10-type phosphorus cast iron. ,3. They 3P [Brake shoes  are obtained by casting from a phosphorus cast iron and are intended for towed rolling stock. Phosphorus gray cast iron in its composition is perlite cast iron with an increased phosphorus content. A phosphorous ternary eutectic system, called steadite, consisting of perlite, cementite, and iron phosphide Fe3P , is form3P .3P), and cementite (Fe3C) [During the solidification of phosphorus cast iron, phosphorus forms ternary phosphorus eutectic, which comprises a solid solution (Fe–C–P), iron phosphide (Fee (Fe3C) ,5. Due te (Fe3C) ,7,8. Thee (Fe3C) ,10,11.The maximum surface occupied by the phosphorus eutectic in cast iron is 15%, and the maximum dimension of its separations is 1 mm. The phosphorus eutectic is characterized by a high hardness 500–600 HB) and high fragility, which explains why it has a great influence on the properties of cast iron. The amount of phosphorus eutectic in cast iron is directly dependent on the phosphorus content, which is also influenced by factors that increase the tendency of phosphorus to segregate ,13. When00 HB andThe presence and increase in the proportion of phosphorous eutectic (a hard constituent) makes the hardness of the cast iron continuously increase. The fragility effect of the phosphorus eutectic is highlighted, in particular, by the fact that the resilience of cast iron decreases continuously with the increase in the phosphorus content .With respect to cast iron, microporosity must be taken into account. This is generally specific to gray cast iron due to contraction microcavities that occur in intercellular spaces but is strongly accentuated in the case of a phosphorus cast iron, when these spaces solidify at low temperatures, resulting in the separations of the phosphorous eutectic ,12,14.Another feature of the eutectic solidification of phosphorus gray cast iron is the presence of phosphorus-rich areas inside eutectic cells in their central part. In such cast irons, the eutectic cells are found “as a spongy material, impregnated with phosphorus-rich liquid” ,8,12,14.-Arched graphite slides of different sizes in an orderly arrangement with very fine blades in the center, fine blades in the intermediate areas, and coarse slides outside the eutectic cells, respectively;-Graphite slides in intercellular spaces, which also belong to neighboring cells;-Separations of the phosphorous eutectic (common ternary) in intercellular spaces;-Phosphorus-rich areas in the central part of the eutectic cells are associated with very fine graphite.Eutectic cells in a phosphorus gray cast iron have the following specific structures ,8,12,14:In general, there are several factors influencing the formation of contraction porosity, specific to the central areas of cast iron parts, including, namely, the nature of the materials used in the load of the elaboration unit, the chemical composition of the cast iron, the thermal elaboration regime, graphitizing modification, and rigidity of forms .For cast iron brake shoes, it is known that at high temperatures during braking (800–900 °C), eutectic phosphide in the immediate vicinity of eutectic cells melts and flows around the austenitic crystals of the rough surface of the shoe. This increases the surface of the wheel contact shoe and develops greater friction ,17,18,19The technical quality conditions and main dimensions of P10 phosphorus cast iron brake shoes for normal gauge railway rolling stock are specified in Task No. 1/SFMR/SDT/2000/Brake Shoes for the motor and towed rolling stock, as approved by the Romanian Railway Authority .Experiments were conducted to study the influence of the chemical composition of cast iron on the hardness of the brake shoes intended for the rolling stock.Experimental research was performed in a foundry that had induction electric furnaces, an automatic air-pressing machine model HFP 2, and a semi-automatic casting line for the manufacture of rolling stock brake shoes. Research was performed on 70 phosphorus cast iron charges, from which 2 types of brake shoes (type S1 and S2) were poured ,21.The process to construct brake shoes consists of obtaining phosphorus cast iron and casting it into shapes, the technological flow of which is composed of the preparation of training mixtures shapes, the elaboration and casting of phosphorus cast iron, and debating and cleaning the brake shoes. The composition of the metal load on the experimental batches was 10–30%, first infused with cast iron, 20–60% cast iron waste (used shoes), and 20–30% steel waste. Dimensional load preparation has a special role in the elaboration process; it must be advanced and prepared. The resultant liquid cast iron was cast by gravity into temporary shapes (the casting temperature varied between 1470 and 1485 °C). A semi-automatic forming–casting line was used, and the casting form included a battery of 8 brake shoes. The cooling of the shoes was performed in the air at the temperature of the foundry. The brake shoes were approved, and the activities of supplying railway products were authorized by the Romanian Railway Authority, AFER. equivalent .Cequiv-Hs and Hd—at one point at each end of the shoe, on its face, after removing 2 mm of material, by grinding or grinding;-Hss, Hsc, and Hsd—at three points located diagonally on the surface of a sample and obtained by cross-sectioning the broken clogs during the shock test.The Brinell method (specified in SR EN ISO 6506-1:2015 ) was useExperimental data on the variation in the hardness of the brake shoes determined both on the surface of the shoe  and, in its section , the hardness measurement points specified in the standard are shown in The data analysis showed that there were small variations in the hardness measured on the side surface and in the cross-section of the brake shoe. This was due to the narrow variation in the chemical composition of the brake shoes. The values of hardness obtained during industrial scale experiments fell within the range of 197–255 HB, which is in accordance with international standards UIC 830-O .The data obtained in industrial practice were processed in the Matlab calculation program (version R2016a). Hardness variation charts were made for carbon, manganese, phosphorus, and silicon for all points where the hardness measurement was performed. Using Matlab software, three-dimensional graphs with regression surfaces and level curves were built for the variation in hardness depending on the chemical composition concerned, and equations of ordinary variation II were generated as follows:In -A microscope with a thickness of G of 100× on polished and not chemically attacked specimens for graphite visualization;-A microscope with G-trushes in the order of 200×, 50 or 100×, 25 or 50× on the same specimens attacked with Nital alcoholic acid solution (4%) to examine the structure with a normal attack time, checking perlite and ferrite and with a prolonged attack duration, to visualize the phosphorus eutectic network.A metallographic structural examination was performed according to the specifications  using an-The variation in independent parameters within the technological limits determines a variation in the dependent parameter with its location on or near a regression surface and in view of dispersion, deviation, and error.-The graphic representations of grade II polynomial functions are surfaces with a stationary point that is either extreme (of the maximum or minimum) or saddle. Their coordinates are within the technological limits of variation for independent parameters, respectively, and within the limits provided by the specifications for the dependent parameter.-The intersection of correlation surfaces with level planes  and level curves were obtained, thus allowing the limits of variation in independent parameters to be established, obtaining a certain value for the dependent parameter, and finding that there are higher variation limits the independent parameters near the stationary point in cases where a saddle point exists. For each graphical representation, the subdomains in which the values for the dependent parameter are found can be indicated, which determines the variation limits for independent parameters.From the equations of the double correlation analysis, as expressed in the analytical and graphic form, a series of conclusions were obtained:-The dependencies presented in -The dependencies presented in -The dependencies presented in -The dependencies presented in -The dependencies presented in -The dependencies presented in By individually analyzing each surface—with the specific influence of two elements on the chemical composition of hardness—the optimal variation intervals of these elements were identified. These were as follows:Taking into account the analysis performed on each diagram, the optimal variation intervals for the analyzed elements resulted in the following: 2.97–3.15%C, 0.53–0.65%Mn, 1.3–1.8%Si, and 0.85–1.02%P. In terms of S content, it varied between 0.08 and 0.09%, and the residual elements did not exceed 0.2%.-A sample  to identify the form of graphite separations, their character and the length of the lamellar graphite separations;-A sample  to determine the perlite configuration and two samples  for the base metal mass and phosphorus eutectic.From each batch, samples were taken and subjected to spectral analysis to determine their chemical composition. The metallographic examination of shoe cast iron was performed according to SR EN ISO 945-1:2019 , and theThe high content of phosphorus improves the friction behavior and wear of this cast iron; the star is formed in the structure. The structure consisting of perlite, graphite, and eutectic phosphorus (steadite) determined the resistance to wear and high fluidity. The properties of the cast iron were determined both by the nature of the metal mass and by the quantity, shape, and dimensions of graphite separations. The properties of resistance and plasticity increased with a reduction in the amount and size of graphite separations and their compaction.-The presence of lamellar graphite separations ;-The proportion of ferrite in the form of isolated islands was 1% and 5% of the area of the samples presented;-A network of uniformly distributed phosphorus eutectic of approximately 15.5% and 12.5% was identified, respectively;-A lamellar perlite configuration, with values of 71% and 72.5%, respectively, was obtained for the presented samples.The analysis of the microstructures presented in In order to verify the results obtained, research was performed on the same technological flow for two charges, pouring S1-type brake shoes from quality cast iron-type P10-phosphorus cast iron.The chemical composition of cast iron is presented in To verify the applicability of the relationships obtained in Matlab and presented in point two, the values of hardness were calculated and compared with those determined in practice and presented in -In -In The Quanta FEG-250 SEM  was used for the micro-structural analysis of samples both with and without a reagent attack. The following microstructures relate to charge A1 and are presented as follows:The microstructural analysis of samples attacked with 5% Nital indicates that the area occupied by perlite was between 45 and 65% (standard structure P50), and the analysis of the degree of perlite dispersion indicated fine slides (standard structure Pi 0.5). The phosphorus eutectic network was evenly distributed for the analyzed samples, and the proportion of ferrite in the form of isolated islands was less than 5% of the sample area.-For the Matlab correlations, we considered, as a dependent parameter, the hardness (determined in two points on the surface of the brake shoe and in three points in the section of the respective brake shoe its average both on the surface and on its section) and the chemical composition of phosphorus cast iron from which the brake shoes were poured, was considered an independent parameter, thus obtaining double correlation equations;-For all the correlations obtained, the correlation coefficient R2 had values of over 0.50, showing that these correlations are significant and adequately reproduce the connection between hardness and chemical composition;-For values of the hardness parameter between 197 and 255 HB, the variation limits for the independent parameters result, namely in C = 2.97–3.15%, Mn = 0.53–0.65%, Si = 1.3–1.8%, and P = 0.85–1.02%. Sulfur varied between 0.08 and 0.09%, and the residual elements did not exceed 0.2%.From the analysis of experimental data taken from industrial practice and processed, the following conclusions were obtained:-From the micro-structural analysis of the two types of clogs, S1 and S2, a distribution of lamellar and very little semi-arched lamellar graphite (type Gl5—maximum 80% and Gl6—minimum 20%) resulted in the form of isolated separations of type Gr1 at no more than 12.5%;-The ferrite present in -The phosphorous eutectic was present on all samples with a lacy appearance and interdendritic separation, the content of which varied between 12.5 and 15.5% for the two microstructures;-The verification of the relations obtained in Matlab with industrial data led to differences of −2.62% and +3.12%, respectively, which can be considered acceptable in the present case;-In the case of the new charges analyzed, the micro-structural analysis was similar; for this batch, it was possible to analyze using high-performance equipment and higher magnification orders.From the analysis of microstructures performed on the two different types of brake shoes, the following conclusions were obtained:


Saliva is a clinically informative biological fluid that contains many biomarkers, allowing multiple analyses to be performed.The objectives of this study were the assessment of the serum and saliva levels of biochemical parameters and intensity of free radical processes in T2DM patients and the identification of the correlation between certain criteria.This case-control study included 40 T2DM patients, which were compared with 40 healthy individuals. The levels of glucose, cholesterol triglycerides, total protein, diene conjugates, and chaperone activity were measured using the spectrophotometric method. The concentration of 8-oxo-2′-deoxyguanosine was assessed by competitive enzyme-linked immunosorbent assay.It was established that the progression of diabetes led to an increase in glucose in saliva. The content of 8-oxo-2′-deoxyguanosine and conjugated dienes increased in serum and this increase was associated with the level of glucose and glycated hemoglobin. The level of protein and chaperone activity increased in the saliva of patients with T2DM compared with the control. The correlation analysis revealed a relationship between total protein concentration and conjugated dienes and between chaperone activity and conjugated dienes in saliva.According to the results of the analysis, the pathological changes in DM affected the salivary glands and their secretions. The obtained results allowed us to recommend using saliva as an alternative to blood for the diagnosis and monitoring of T2DM treatments since it is readily available and quickly responds to changes in metabolism in the body. Diabetes mellitus (DM) is one of the most common diseases affecting more than 463 million people in the world . In the Diagnosis of diabetes, as well as regular monitoring of compensation for the disease, is accompanied by constant blood sampling for analysis. Now, the search for new non-invasive methods for detecting diabetes is being carried out. Saliva is one of the promising targets for diagnosing DM.Saliva is an accessible biomaterial providing a link between the external and internal environment of the body. The salivary glands selectively carry out the transport of substances from the blood plasma into the saliva, have an endocrine function, and respond to any changes in the state of internal organs and body systems .Many researchers believe that a change in the composition of the saliva of the large salivary glands can be a marker of a pathological process in the body . MetabolHuman saliva is a unique biological fluid, the change in biochemical parameters of which reflects the state of metabolic processes in the body and has clinical and diagnostic value . The useHeat shock proteins (HSP) involved in the regulation of growth, development, signaling, cell death, and other physiological processes, were found in mixed saliva . Express8-Oxo-2′-deoxyguanosine (8-oxo-dG) is a well-known biomarker of oxidative stress and associated diseases; it is determined in various tissues and body fluids: blood, urine, saliva, and liver . Many stAn increased concentration of glucose has a negative effect on the resistance of capillaries and increases the permeability of blood vessels in the oral cavity, which in turn affects the biochemical composition of saliva, in particular, contributes to the development of free radical oxidation, including lipid peroxidation (LPO) as a result of damage to biological membranes . In thisThis case–control study was conducted at the endocrinology department of the BUZ VO “Voronezh Regional Clinical Center for Specialized Types of Medical Care” Russian Federation. The diagnosis of T2DM was established in accordance with the classification criteria, according to clinical guidelines . The resThis study included 40 patients with T2DM (20 men and 20 women). The control group consisted of 40 apparently healthy individuals with normal indicators of general and biochemical blood tests. The exclusion criteria from the study were: periodontitis, viral hepatitis, acute infectious diseases, acute myocardial infarction, malignant neoplasms, and cerebrovascular accident.The collection of mixed saliva was carried out in the morning on an empty stomach after rinsing the mouth with water using a saliva collection kit (Sarstedt D-51588 Numbrecht), the cotton swab for saliva collection was kept in the mouth for 10 min. During the collection of saliva, the donors sat, did not speak, and breathed through their nose. A clear saliva sample for use in the study was obtained after centrifugation for 10 min at 1400 g.Quantitative determination of saliva glucose levels was performed by the glucose oxidase method. The absorbance measurements were carried out at 510 nm. The protein concentration of saliva samples was determined using the biuret reaction .The chaperone activity of mixed saliva was determined using a model test system based on the suppression of insulin aggregation. The kinetics of insulin aggregation induced by dithiothreitol and a temperature of 40°C were measured by turbidimetric assay. Aggregation was recorded using a Spekol 100 spectrocolorimeter with a thermostated cuvette and an automatic recorder at a wavelength of 430 nm .The determination of the concentration of 8-oxo-2′-deoxyguanosine in saliva was carried out by competitive enzyme-linked immunosorbent assay using an HT 8-oxo-dG ELISA Kit ; the registration was carried out at a wavelength of 450 nm.Determination of the concentration of diene conjugates (DCs) in mixed saliva was performed using the spectrophotometric method based on the appearance of conjugated double bonds in polyunsaturated fatty acid molecules during LPO. The registration was performed at 233 nm.The blood glucose level was assessed using a Satellite Plus glucometer .Statistical data processing was performed by the methods of mathematical and medical statistics using the Microsoft Office Excel data analysis package and the STADIA 7.0 statistical package . Quantitative data are presented as M ± m, with M− sample mean, m − standard error of the mean. For the identification of significant differences between independent groups, a Student’s t-test was used. For testing statistical hypotheses, a 5% significance level was used.Pearson’s correlation coefficient was used to assess the correlation of the studied parameters of blood serum and mixed saliva.During the study, saliva and blood serum samples were obtained from 40 people with T2DM. The clinical parameters of study participants are presented in In the group of apparently healthy individuals, the glucose level in the blood serum was 4.3 mM in men and 4.1 mM in women, which correspond to acceptable values [There was also an increased content of glycated hemoglobin in the examined patients by 1.6 times compared to the control values (p < 0.05) in men, and by 1.7 times (p < 0.05) in women. The concentration of glucose in saliva in patients with T2DM compared with the control group was increased by 3.5 times (p < 0.05) in men and 3.4 times (p < 0.05) in women, which is consistent with the data on the degree of hyperglycemia in the blood .An increase in the concentration of cholesterol and triglycerides in both blood serum and saliva was revealed in patients with T2DM. At the same time, a positive correlation between the content of total cholesterol in saliva and blood serum was observed . A high positive correlation between the concentration of triglycerides in the blood serum and saliva in patients with T2DM was identified .The results of the study demonstrated a threefold increase in the content of 8-oxo-dG in the mixed saliva of patients with T2DM in both men and women in comparison with apparently healthy individuals . The conxy = 0.498) and women (pxy = 0.387) with T2DM. Associations between the concentration of 8-oxo-dG and glycated hemoglobin (pxy = 0.535) and between the content of 8-oxo-dG and the concentration of glucose in the blood (pxy = 0.534) were also detected.The dependence of the intensity of free radical processes on the level of glycaemia was confirmed by the data of correlation analysis between the concentration of glucose in saliva and the concentration of DC in men (pxy = 0.347) and between the concentration of DC and chaperone activity of saliva in both men (pxy = 0.313) and women (pxy = 0.327) with T2DM were found.As our studies have shown, the concentration of total protein in the saliva of patients with T2DM was increased on average by 29.4% (p < 0.05) in men and by 33.4% (p < 0.05) in women compared with the control group . An incrxy = 0.314) and DC concentrations (pxy = 0.414) in women.It should be noted that data reflecting the correlation relationship between the age of patients with T2DM and biochemical parameters in saliva were obtained. Thus, positive correlations were found between age and glucose  and hexosamine pathways, autooxidation during DM is known. According to the literature, an increased glucose content in saliva leads to the accumulation of end products of glycation, contributing to an increase in the production of reactive molecules damaging biological molecules, which was reflected in an increase in the level of DC and 8-oxo-dG ,21. The It is known that most salivary proteins originate from the salivary glands, but in various diseases, including T2DM, they can have a different origin. Associated, for example, with an inflammatory process or tissue damage. Probably, developing oxidative stress is one of the factors involved in tissue damage, which was confirmed by correlation analysis. In addition, the end products of glycation formed during T2DM are able to interact with monocytes, promoting their activation, which is characterized by the induction of migration and the release of proinflammatory cytokines .These cytokines promote tissue damage primarily by enhancing the inflammatory activation of T-cells as well as facilitating neutrophil degranulation. Probably, an increase in the concentration of total protein and chaperone activity in saliva in patients with T2DM was associated with hyperglycemia and the development of oxidative stress, which was confirmed by the results of correlation analysis.The revealed correlations between the DC level and the chaperone activity of saliva were probably due to the activation of HSP synthesis under the influence of ROS . In addiThe analysis of the correlations between the age of patients and the biochemical parameters of saliva indicates that T2DM belongs to diseases associated with aging, in the pathogenesis of which the development of oxidative stress plays an important role.The obtained results allowed us to recommend saliva as an alternative to blood for the diagnosis and monitoring of T2DM treatment, since this biological fluid is readily available and quickly responds to changes in metabolism in the body. The method of collecting saliva is painless and atraumatic, which helps to reduce the risk of infection for patients and medical personnel, and also allows multiple sampling for research.


Routine use of the script concordance test (SCT) is not common in Brazilian universities. This study aimed to analyze application of the SCT in the medical school of a Brazilian university. Quantitative, analytical and descriptive study in the medical school of a Brazilian university. A total of 159/550 students participated. The test comprised ten clinical cases within internal medicine, with five items per case, rated on a five-point Likert scale. The test was scored in accordance with a marking key that had been validated by a reference panel. In the pre-clinical and clinical phases, the mean scores were 51.6% and 63.4% of the maximum possible scores, respectively. Comparison of the means of the responses among all the years showed that there were significant differences in 40% of the items. The panel marked all the possible answers in five items, while in one item, all the panelists marked a single answer. Cronbach’s alpha was 0.64. The results indicated that the more senior students performed better. Construction of an SCT with discriminative questions was not easy. The low reliability index may have occurred due to: a) problems with the construction of the questions; b) limitations of the reference panel; and/or c) the scoring key. This instrument is very difficult to construct, apply and correct. These difficulties may make application of an SCT as an assessment method unfeasible in units with limited resources. This study was approved by the UFG Ethics Committee under number 176/12.All 550 students from the first to the fifth year of the undergraduate program were invited to take an SCT. The recruitment of participants took place at FM-UFG by means of an invitation to all students who met the inclusion criteria. Students over 18 years of age who were enrolled and who signed the consent form were included. Students whose native language was not Portuguese and sixth-year students were excluded. The latter were excluded because of difficulty in accessing groups that were attending training courses outside of the institution.The SCT was applied on previously scheduled days, on one day for each year of the program, on the FM-UFG premises. The test was completed by each student over a 90-minute period, regardless of year, and was the same for all the study subjects.The test was developed by the researchers using 10 short clinical cases within internal medicine involving diagnostic questions on the following topics: acute myocardial infarction, pleural effusion, pneumonia, pulmonary embolism, malignant stomach tumor, cirrhosis of the liver, gastroesophageal reflux disease, congestive heart failure, acute renal failure and secondary hyperparathyroidism. There were five items for each of the ten clinical cases, thus yielding a total of 50 items. The clinical scenarios were followed by a series of questions, which were presented in three parts. The first part (“if you were thinking...”) contained a relevant diagnosis. The second part (“and then you find...”) presented a new clinical finding, such as a physical sign, a pre-existing condition, an imaging study or laboratory test result. The third part (“this option would become...”) displayed an answer in a Likert scale format, in which the subject quantified how much influence the new information (second part of the question) would have on what he was thinking (first part of the question). The answers followed a five-point Likert scale that captured the participants’ decisions .2The students had to decide what effect the new discovery (second part of the question) had on the state of the option cited in the directions (first part of the question), whether positive, negative or neutral, and the intensity of this effect using the Likert scale. The Likert scale structure was the same for the entire test, with negative values on the left, 0 in the neutral position and positive values on the right  .The students were instructed not to answer questions for which they did not understand the answers, and these questions were given a score of 0 in the overall rating.The test was scored in accordance with a marking key that had been validated by a reference panel made up of 10 professors of the FM-UFG Department of Clinical Medicine. The professors were invited to volunteer because they were experts in various areas of medical knowledge and belonged to the FM-UFG Department of Clinical Medicine. Three were cardiologists; two were nephrologists; one was a pulmonologist; one was a hematologist; one was a gastroenterologist; one was from the Intensive Care Unit; and one was from the Immunology Department. Scoring the SCT involved comparing the responses provided by the participants with those of the reference panel. The panelists were asked to answer the test individually, and their answers were used to develop a scoring key. For each response, the score was the number of panel members who chose this response. Therefore, for every reference panel response to each question, a score of 0.1 was awarded, creating a maximum score of 1.0 for each question .The Microsoft Excel 2007 software was used to tabulate the data, and the statistical analysis was performed using the SPSS for Windows software, version 16.0. A value of 5% was used as the significance level (P < 0.05). The Kruskal-Wallis test was used to compare the variable of school year in relation to the score for each question. The overall Cronbach’s alpha was calculated to evaluate the internal consistency among the questions.Out of the total of 550 students (110 per school year from the first to fifth year), 159 participated in the study. There were 27/110 (24.5%) of the first-year students, 39/110 (35.5%) of the second-year students, 30/110 (27.3%) of the third-year students, 27/110 (24 5%) of the fourth-year students and 36/110 (32.7%) of the fifth-year students took the SCT, thus yielding the total of 159, or 28.9% of the students.The reference panel shown in pre-clinical, i.e. those in the first and second years, who have no contact with patients; and clinical, i.e. those in the third, fourth and fifth years, who were already interacting with patients. Students in the pre-clinical phase obtained a mean of 13.94 points or 51.6% of the maximum score, and those in the clinical phase obtained a mean of 17.11 points or 63.4% of the maximum score.The students were grouped as Through completion of the Kruskal-Wallis test, which compared the means of the responses to each question and its items among students of all years, significant differences were only found in the following 20/50 items (40%): question 1 items 2, 3 and 5; question 2 items 1, 3, 4 and 5; question 3 items 1, 4 and 5; question 4 items 1 and 5; question 5 items 4 and 5; question 7 item 1; question 9 items 1, 3 and 5; and question 10 items 3 and 5.The expert panel chose all the possible answers in question 3 item 3, question 5 item 2, question 6 item 4, question 9 item 2 and question 10 item 5. Additionally, the entire expert panel chose the same response in question 7 item 2.The SCT instrument in this study had a reliability index  of 0.64.,,,,,,,,In this study, 159/550 (28.9%) of the FM-UFG students took the test. This number corresponds to what has been found in other studies.In the present study, the SCT was applied to students at different stages of the medicine program at FM-UFG, from the pre-clinical phase (first and second years) to the clinical phase . Students in the pre-clinical phase obtained a mean of 51.6% of the maximum score, and those in the clinical phase obtained a mean of 63.4% of the maximum score. There are few studies on the SCT with students in the pre-clinical phase.,Comparing the mean scores of the students in the pre-clinical phase with those of the students in the clinical phase, an expected difference in student performance can be seen, with the senior students performing better. The SCT assumes that the individual examined interprets the data presented.,The SCT was able to demonstrate that students in the pre-clinical and clinical phases had different levels of performance according to the degree of knowledge and maturity expected at each stage.,Problems with SCT construction regarding several questions were identified in the present study. Only 40% of the items were able to differentiate between the students in the clinical and pre-clinical phases. In question 3 item 3, question 5 item 2, question 6 item 4, question 7 item 2, question 9 item 2 and question 10 item 5, there was very low discrimination. There were no significant differences among the five years of the program with these questions. Construction of an SCT with discriminative questions regarding clinical reasoning is not easily achieved.In our test, in question 7 item 2, all the panelists chose the same answer . Questio,In question 3 item 3, question 5 item 2, question 6 item 4, question 9 item 2 and question 10 item 5, the panel chose all possible answers ; thus, t,,The SCT instrument in this study had a reliability index the problems cited regarding the construction of the questions;a reference panel of 10 professors, which is below the acceptable minimum;applying an SCT based solely on diagnostic ability in internal medicine with volunteer professors from the FM-UFG Department of Clinical Medicine; these volunteers from various areas of knowledge were required to answer questions and items outside their area of specialized knowledge , although all the panelists had knowledge of internal medicine; this type of panel may thus have caused some distortion in the reference panel answers; and,,using a five-point Likert scale.In smaller academic units with limited resources, it may be more efficient to replace the aggregate five-point scoring methodologyIn medical education practice, all assessment instruments have limitations.Regarding the limitations of this study, this test was the first SCT developed by our group. Due to local conditions, no pilot study (which might have overcome some of the difficulties) was conducted. The FM-UFG Department of Clinical Medicine has 42 professors, and 10 volunteered for the reference panel: this number was shown to be limited with regard to assessing various questions that were outside the panelists’ area of expertise. A traditional five-point Likert scale was selected for the SCT. However, we believe that these limitations do not invalidate the process developed and that the observations made in this study can help researchers and educators who plan to conduct the SCT.The SCT was able to demonstrate that students in the pre-clinical and clinical phases had different levels of performance according to the degree of knowledge and maturity expected at each stage.This instrument is very difficult to construct, apply and score. These factors may make the application of the SCT as an assessment method unfeasible in units with limited resources.


The novel multiplex detection method, which can increase accuracy and improve the ability of comprehensive analysis, can basically meet the requirement of detection and the diagnosis of the proteomics of nutrition and health.We attempted to construct and evaluate a novel detection method to realize simultaneous detection based on a multiplex liquid protein chip technique for nine nutrition-and-health-related protein markers to meet the requirement of an accurate, simultaneous and comprehensive analysis of the proteomics of nutrition and health. The lower limits of detection, biological limits of detection and regression equations of serum ferritin (SF), soluble transferrin receptor (sTfR), c-reactive protein (CRP), retinol-binding protein4 (RBP4), apolipoprotein B (ApoB), alpha-fetoprotein (AFP), prealbumin (PA), carcino-embryonic antigen (CEA) and D-Dimmer (D-D) were determined after a series of optimal experiments. Then, the results of the methodological evaluation for this novel method indicated that the accuracies were between 70.12% and 127.07%, the within-run precisions were between 0.85% and 7.31%, the between-run precisions were between 3.53% and 19.07%, the correlation coefficients between this method and other methods were above 0.504 ( The detections of protein markers of nutrition and health are necessary when evaluating the statuses of nutrition and health ,2, whichThe statuses of the protein, Vitamin A, and iron, as well as cardiovascular, cerebrovascular disease and carcinoma, are five main fields of nutrition and health in China , which mAt present, immunological methods are mainstream ways of detecting the above nine protein markers, which mainly include electro-chemiluminescence immunoassay (ECLIA), radioimmunoassay (RIA), immune turbidimetry, and the enzyme-linked immunosorbent assay (ELISA). They have once appeared with a favorable performance in the previous study at a certain degree, while all of them could not realize multiplex and simultaneous detection for the above nine protein markers. So, the detection efficiency and system error of these conventional methods could not meet the requirements of nutrition-and-health proteomics on a more accurate and panoramic analysis for the above five main fields.The protein chip technique is an important detection means that is often used to support the data analysis of proteomics, and it is one kind of multiplex and quantitative detection technique which could realize the simultaneous detection of multi-protein markers using the same one detection. According to the different detection conditions, the protein chip includes a solid chip and a liquid chip. The basic detection principle of the protein chip is the known different protein molecules  are, respectively, which are fixed on the surface of the carrier (solid surface or microsphere) of the protein chip after the carrier is processed as a special chemical method. Then, these protein molecules capture the corresponding target proteins (protein markers) in serum, plasma, lymph, urine and secretion according to the immunological characteristics of these known protein molecules. After that, the quantitative results of these protein markers are used to conduct proteomics analysis. Until now, the protein chip technique has been widely used in many fields of clinical detection, such as the screening of gene expression, the detection of a specific antigen or antibody, the study of the interaction between different proteins, and the research for new medicine . More anOur team constructed multiplex detection methods for protein markers by using solid protein chips in two aspects of the nutritional field. These two explorations aimed to identify the simultaneous detection of SF and sTfR in the serum  and the The liquid protein chip is a new-generation biochip and a new type of research platform for protein markers, which can provide technical support for multi-analyte profiling (MAP) immunoassay. In the late 1990s, scientists invented MAP technology, which was a major advance in multiplexed biological assays. This technology drew from the strengths of solid-phase separation technology without the typical limitations of solid-phase reaction kinetics. By combining advanced fluidics, optics, solid chip, and digital signal processing with proprietary microsphere technology, MAP technology enabled a high degree of multiplexing within a single sample volume , which cWhether the liquid protein chip technique can be used in the detections of the above nine protein markers of nutrition and health to provide a more accurate and comprehensive analysis for proteomics of nutrition and health remains to be seen. The current references do not indicate that any research has attempted to construct a similar method that can simultaneously detect the above nine proteins until now.Depending on the above analysis and hypothesis, our team adopted the timely liquid protein chip technique to construct a new multiplex detection method for nine protein markers of nutrition and health so as to overcome the limitation of the solid protein chip and to further increase the detective throughput of protein markers in nutrition and health proteomics.According to our research, we attempted to introduce the construction and evaluation of a novel MAP capture sandwich immunoassay for nine nutrition-and-health-related protein markers based on the liquid protein chip technique in this article, which could provide better technique supports and more comprehensive data for the analysis of the proteomics of nutrition and health in the future. This attempt is a meaningful exploration of the liquid protein chip technique in nutrition and health. Further, we also hope that the nutrition-and-health proteomics based on this constructed method can play more roles in the monitoring and intervention of nutrition and health of large-scale crowds in the future.All the reagents of this research can be searched from MILLIPLEX Multiplexing Assays ; Millipore-Q Academic ; Allegra x-22R Centrifuge ; SpectraMax I3X Enzyme marker ; Desk centrifuge 5418 ; Vortex mixing device ORTEx Genius ; Digital ultrasonic cleaner ; Bio-Plex Handheld Magnetic Washer ; Oscillator ; Magnetic Shelf DvnaMag-2 ; Biochemical incubator SPX-150B ; 7600 Series Automatic Analyzer ; Roche E601 Immunoluminescence instrument .The serum samples were obtained from the UNICEF project “Observation on the YYB intervention on health, cognition and behavior”. All guardians agreed with the content of informed consent from the National Institute for Nutrition and Health (NINH) and Chinese Center for Disease Prevention and Control, and the number of NINH ethic committee was (NO. 2018-20). All serum samples were frozen at −80 °C before they were detected.The basic operation procedures of the coupling of the microsphere and the capture of antibodies include the activation of the microsphere with a ProteOn Amine Coupling kit, carbodiimide coupling for antibodies with a Bio-Plex Amine Coupling kit, blocking with a Bio-Plex Amine Coupling kit, and storage with a Bio-Plex Amine Coupling kit. All of these operations were implemented according to the introductions of the ProteOn Amine Coupling kit and Bio-Plex Amine Coupling kit. Additionally, the coupled microspheres with different kinds of capture antibodies were stored at 4 °C.The basic operation procedures where the antigen combines with a capture antibody include the addition of the coupled microspheres in each well of 96 well bottom plates (3000 microspheres in each well), the rinse of microspheres , the addition of antigens (standard substance or serum) to each well, and horizontal oscillation with 700 rpm (1 h) after 1100 rpm (30 s) (Room temperature and avoid light).The basic operation procedures where the antigen combines with the detection antibody with biotin include the rinsing of microspheres , the addition of a detection antibody with biotin into each well, and a horizontal oscillation with 700 rpm (30 min) after 1100 rpm (30 s) (at room temperature and avoid light). The basic operation procedures for the detection of the antibody combined with SAPE include the rinsing of microspheres , the addition of 50 μL SAPE in each well, and horizontal oscillation with 700 rpm (15 m) after 1100 rpm (30 s) (at room temperature and avoid light). Additionally, then, microspheres were re-suspended under the condition of 700 rpm at 15–20 min after 1100 rpm (30 s) (at room temperature and avoid light).Finally, 9 kinds of antigens that, respectively, combined with the corresponding microsphere can simultaneously be detected, and their signal values could be simultaneously obtained under 532 nm and 635 nm through MILLIPLEX Multiplexing Assays. The chessboard titration analysis was adopted to optimize the appropriate concentration of the capture antibody and the titer of the detection antibody of each protein marker. In this method, the optimal concentration of the capture antibody for each protein marker was determined from 3 alternative concentrations  according to the pre-experiment of each capture antibody, and the optimal titer of the detection antibody of each protein marker was determined from 3 alternative titers  according to the suggestion of the manufacturer for each detection antibody, and the concentrations of each protein marker included high and low concentrations  according to the normal range and pre-experiment of each protein marker, and 1% BSA was used as a blank control for each protein marker. The specific operation refers to The optimal combination of the capture antibody concentration and detection antibody titer was determined when both the highest signal value and lowest background value simultaneously met under this combination, and the distance value between the high and low concentration of the protein marker was the biggest at this time.The cross-reaction experiment was adopted to verify the specific binding ability between the antigen and antibody ,6. The cp < 0.05). There is a cross-reaction between one antigen and another antibody  if the cross-reaction rate of them is larger or equal to 20% of that between this antigen and the antibody itself . The cross-reaction rate can be calculated using Formula (1):There is a specific binding ability between the antigen and antibody if the detection result is significantly bigger than that of the blank control  , and theThe corresponding concentration value of the twofold standard deviation (2SD) of the signal value of a blank well is the Lower Limit of Detection (LLD), and the concentration value is the Biologic Limit of Detection (BLD) when it is just larger than LLD after it subtracts 2SD.The mixed standard substance of 9 protein markers was successively diluted 20 times using serial 2 multiple dilutions, which meant that the mixed standard substance that included SF, sTfR, CRP, RBP4, ApoB, AFP, PA, CEA and D-D was successively diluted from a high mixed point  to a low mixed point . The concentration of the capture antibody and the titer of the detection antibody for each protein marker depended on the optimal results of X-axis and Y-axis, respectively, and then, the S-curve of each protein marker could be drawn according to the relationship between the concentration and signal value.The concentration and the signal value of each protein marker in the mixed system appeared on the S-curve of each protein marker. Then, the mixed standard substance of SF, sTfR, CRP, RBP4, ApoB, AFP, PA, CEA and D-D was successively diluted from high mixed point  to low mixed point  as doubling dilution. At last, 9 standard regression equations for the 9 protein markers of the mixed system were simultaneously established depending on the above 6 standard points of each protein marker.After that, the 6 standard points of each protein marker in the mixed system were determined according to the range of the straight linear of the corresponding The determination coefficient of each regression equation should be bigger than 0.95 at least.The adding standard recovery was used to evaluate accuracy according to the “Guidance on the Verification of Quantitative Measurement Procedures used in the Clinical Chemistry , abbreviation is Guidance”. Two random serums were diluted, respectively at 1:40 (serum I) and 1:100 (serum II), and the mixed antigen standard  was added, respectively, into serum I and serum II as the 4% of basic serum volume (50 μL) and 5% of basic serum volume (50 μL). The standard curve of each protein marker refers to The adding standard recovery rate was calculated through Formula (2).R: adding standard recovery, V: the volume after standard was added in basic serum, V0: the volume of basic serum, C: the detected concentration after standard was added in basic serum, C0: the detected concentration of basic serum, Cs: the concentration of standard of each protein marker.The 100.0% ± 30.0 is an appropriate accuracy range for adding a standard recovery of each protein marker of the mixed system .The precision evaluation included within-run precision and between-run precision. The operations refer to “Guidance”. Two random serums were diluted after at 1:60 and were, respectively, detected with 10 replicates in one day in order to conduct the analysis of within-run precision for this novel method. Another two random serums, which were used to perform a between-run precision, were stored at −20 °C after they were, respectively, divided into 6 aliquot tubes, and freeze–thaw cycles should be avoided. The whole experiment period of between-run precision was 6 days, and 2 serums for each day were, respectively, diluted at 1:60, and they were, respectively, detected with 3 replicates. The standard curve of each protein marker refers to The coefficient of variation (CV) in of the detection results of each protein marker was adopted to evaluate the precision of this method. The within-run precision and the between-run precision of this method should be, respectively less than 10% and 20%.The operation refers to “Guidance”. A total of 16 random serum samples were, respectively, aliquot into 10 groups, and one group was used to detect 9 protein markers simultaneously through one detection using the liquid protein chip, and four groups were used to detect sTfR, CRP, ApoB and PA, respectively, in four separate experiments through the use of the ELISA method, and 3 groups were used to detect respectively SF, RBP4 and D-D, respectively, in 3 separate experiments using the immunoturbidimetry method, and 2 groups were used to detect AFP and CEA, respectively, in 2 separate experiment using the ECLIA method.The 16 serum samples, which were used to detect 9 protein markers simultaneously in one detection using the liquid protein chip, were detected after they were diluted at 1:60, and the standard curve of each protein marker refers to p < 0.05), while the result of the pair comparison between two different methods should not be significant (p > 0.05).For 9 protein markers of the same 16 serum samples, the detected results using a liquid protein chip were compared, respectively, with that of using a separate detection method. The result of the correlation analysis between two different methods should be significant (The operation refers to “Guidance”. Direct bilirubin (DBIL), indirect bilirubin (IBIL), hemoglobin (Hb) and triglycerides (TG) were adopted to judge whether the detection results of the liquid protein chip could be interfered by jaundice, hemolysis and lipoidemia.Two random serum samples were aliquoted into 9 groups after they were diluted as 1:60. The 2 diluted serum samples of the first group (control group) were used to directly detect the concentrations of 9 protein markers, and those of the second and third group (jaundice I group) were used to detect the concentrations of 9 protein markers after they were added, respectively, into high and low concentration DBIL . Those of the fourth and fifth groups (jaundice II group) were used to detect the concentrations of 9 protein markers after they were added, respectively, into high- and low-concentration IBIL . Those of the sixth and seventh groups (hemolysis group) were used to detect respectively the concentrations of 9 protein markers after they added interfered, respectively, into high and low concentration Hb , and those of the eighth and ninth group (lipoidemia group) were used to detect, respectively, the concentrations of 9 protein markers after they were added, respectively, into high and low concentration TG . The dilution of 1% BSA was used, and each case was repeated 3 times. The standard curve of each protein marker refers to The mean values for the concentration of the control group and other groups were analyzed, respectively, and the difference value between each experiment group and control group were further calculated. Additionally, the interference rate was calculated by relative bias as Formula (3).I: interference rate, D: the difference value of mean values between each experiment group and control group, C: the mean value of control groupThe intervention would affect the analytical specificity of liquid protein chip if the interference rate was larger than or equal to 20%.t-test or Wilcoxon matching pair symbol rank sum test and the correlation analysis were adopted, respectively, to conduct a comparison with the reference method, and the correlation coefficient was analyzed by a t-test (p < 0.05). Statistical graphs were produced with Microsoft Excel 2010 .SPSS19.0  was used for statistical analysis. In the research, at least three replicated tests were performed in the liquid protein chip. All the data were presented as the means ± standard deviation (SD). The least square method was used to construct the regression equation, and the paired The result of the optimal combination of the capture antibody concentration and the detection antibody titer for each protein marker indicated that the optimal concentration of the capture antibody and the optimal titer of the detection antibody of SF were 53.3 μg/mL and 1:100, those of sTfR were 53.3 μg/mL and 1:400, those of CRP were 26.6 μg/mL and 1:1000, those of RBP4 were 53.3 μg/mL and 1:1000, those of ApoB were 80.0 μg/mL and 1:400, those of AFP was 53.3 μg/mL and 1:100, those of PA were 26.6 μg/mL and 1:200, those of CEA was 80.0 μg/mL and 1:200, and those of D-D were 80.0 μg/mL and 1:200, respectively.The detection conditions of the subsequent experiment depended on the parameters of the above optimal combination.The cross-reaction rates of each protein marker and different antibodies appear in The LLD and BLD of each protein marker are shown in The S-curves of nine protein markers, which were simultaneously detected by using the liquid protein chip in the same detection, displayed in The regression equations and determination coefficients for the nine protein markers that were simultaneously detected using a liquid protein chip in the same detection are in The detection method of the liquid protein chip could simultaneously detect nine protein markers in the same detection, which had been constructed until now.The accuracy range of the nine protein markers was between 70.12 and 127.07%, and the accuracy of each protein marker is shown in The precision ranges of the within-run precision and between-run precision were, respectively, 0.85–7.31% and 3.53–19.07%. The specific results are shown in p < 0.05) and are shown in The results indicate that the correlation coefficient of each protein marker between the liquid protein chip and another corresponding detection method was significant (p > 0.05), which are shown in In addition, the results of the pair comparisons indicated that there was no significant difference between any of the liquid protein chips and other corresponding detection methods (The results indicate that a low concentration of DBIL (28.00 nmol/L) and a high and low concentration of IBIL  would not interfere with the detection results of nine protein markers. In addition, the high concentration of DBIL (5.70 μmol/L), the high and low concentration of TG , and the high and low concentration of Hb  could interfere with the detection results of the liquid protein chip to a certain of degree. The liquid protein chip is a core technique of proteomics, which can provide technical support for the systematic research of a field when different protein indicators are needed to explain a phenomenon . Our resAs we know, SF, sTfR and CRP ,24,25 caThe cross-reaction rate could directly affect the accuracy and recovery rate of the protein marker. Although it is very important for accuracy, there is not a uniform and mature threshold value standard in the field of liquid protein detection. Therefore, we had no choice but to set 10% and 20% as the relative threshold values of the cross-reaction rate according to the requirement of “Guidance on the Verification” on accuracy and the results of our team on other similar research . ConsideThe LLD results indicate that the LLDs of liquid protein chip on eight protein markers  were, respectively, lower than those of the comparison methods, which appeared in the preponderance of the liquid protein chip for the detection of these indicators. Our team had constructed a method for the solid protein chip to detect SF and sTfR simultaneously, and the comparative results indicated that the LLDs of the liquid protein chip were obviously better than those of the solid protein chip when simultaneously detecting SF and sTfR, which further proved that the performance of liquid protein chip was better than that of solid protein chip. The comparisons among the above different methods can be found in Referred to as “Guidance on the Verification of Quantitative Measurement Procedures used in the Clinical Chemistry ”, we preliminarily completed the evaluation of the new detection method from four aspects, including accuracy, precision, a comparison with the reference method, and analytic specificity. For the accuracy of this method, the evaluated result indicated that it met the basic requirements of our research at the preliminary stage, especially at the early application stage of the new technique, because this method took into consideration all factors of the nine protein markers and realized the synergy of detecting nine protein markers in the same detection; additionally, the collection of comprehensive and systematic information in one detection is more important for the research of nutrition-and-health proteomics. In the evaluation of accuracy, the recovery rates of CRP, AFP, PA and CEA exceeded 100%, which reasons that the operation errors of adding standard substances are larger or the specificities of the antibodies are relatively weak. Therefore, we need to further control the operation of experimenters and choose better antibodies with better specificity in the next stage of research so as to improve the recovery rate.In comparison with the reference method, the reason that we did not choose the same method as the reference methods for detecting different protein markers is there is no golden standard in the field of detecting protein, so we had to adopt the common method of aiming at each protein marker as a reference method for each protein marker. Therefore, ELISA was used as a comparison method for the liquid protein chip to detect sTfR, CRP, ApoB and PA, Immunoturbidimetry was used as a comparison method for the detection of SF, RBP4 and D-D, and ECLIA was used as comparison methods to detect AFP and CEA according to the practical conditions of our routine detections. In addition, although eight samples were enough according to the requirement of the comparison within the reference method of “Guidance on the Verification of Quantitative Measurement Procedures used in the Clinical Chemistry ”, we still randomly selected 16 samples to perform this evaluation. The reason for this is that we wanted to verify the feasibility of our method as fully as possible. The results indicate that our method was competent in the comparison of each conventional method. Although the diagnostic efficiency of our method is equal to single detection, our method is able to produce more comprehensive information that could contribute to analyzing the relationship among these indicators from the viewpoint of proteomics. In addition, we also can provide support to nutrition-and-health proteomics by using this method so as to explore the relationship between nutrition and health in the next stage of our research.In the evaluation of analytical specificity, the results indicate that the sample should not be icterus, lipoidemia and hemolysis at a certain degree, which means that the detection result of our method might be disturbed by some disease factors. In the next stage, we need to further improve our method by optimizing the detection condition so as to improve the applicability of this method.At present, more and more researchers are paying more and more attention to the detection of nutrition- and health-related protein markers. Our detection method realized the simultaneous detection of nine protein markers of each of the 40 samples within 5 h, which could not only decline a system error  but coulFor the comparison of multiplex and single plex protein detection, the core of the controversy is actually the diagnostic value, which is appropriate between combined diagnosis on the basis of combined detection and the combined diagnosis on the basis of single detection for the same sample. Referring to the discussion of accuracy above, our research has proved that the diagnostic value of the combined diagnosis on the basis of combined detection (multiplex detection) is equal to that of the basis of single detection, while the results of multiplex detection could provide more comprehensive and accurate information for nutrition-and-health proteomics in one detection.On the other hand, the detective throughput is one key point of the comparison between different multiplex detection methods, which directly influences the overall performance of a multiplex detection method. The throughput of different research is generally different, which depends on the purpose of each research. At the present, although the theoretic throughput of the MAP immunoassay technology can reach 50 protein markers , the ranWe hope our novel MAP immunoassay method can gradually be used in the comprehensive assessment of nutrition and health and the monitoring of the large-scale population so as to provide technical support for real-time and effective prevention or for the accurate intervention of nutrition and health.Based on the liquid protein chip technique, a novel multiplex detection method for nine nutrition-and-health-related protein markers was successfully constructed after we determined the optimal combination of the concentrations of the capture antibody and detection antibody for each protein marker, determined the cross-reaction rate of each protein marker, and determined the LLD and BLD of each protein marker through a series of optimal experiments. At the same time, the evaluated results of the accuracy, precision, and comparison with the reference method and analytical specificity have indicated that this novel method can basically meet the requirement of the laboratory and can preliminarily replace conventional methods. Based on the above analysis and discussion, the constructed novel method based on a liquid protein chip is a worthy exploration in the fields of nutrition and health, which might provide more support for nutrition-and-health proteomics, and might be used gradually in the comprehensive assessment of nutrition and the health of large-scale populations in the future.