An enzyme immunoanalytical system based on sequential cross-flow chromatography

A new enzyme immunoanalytical concept that can be used for point-of-care testing has been investigated. Enzyme as a tracer requires a separate reaction step for signal generation, which follows the completion of immune complex formation with analyte (e.g., Hepatitis B surface antigen) in a sample. This has been a major factor limiting its utilization within the laboratory. We carried out such sequential processes employing chromatographic analysis, using two crosswise-arranged membrane pads in vertical and horizontal directions. The vertically arranged pads were the same as those in the usual format for pregnancy testing, for instance, with the exception of the use of horseradish peroxidase (HRP) as tracer. By placing the horizontally arranged pads on each lateral side of the signal generation pad in the vertical arrangement, they were employed to supply substrate to the enzyme present in the immune complexes. The substrate flow was initiated after the antigen-antibody bindings to produce a signal, which was typically a color change in proportion to the analyte concentration. Under optimal conditions, the use of HRP labeling increased the detection capability of the assay approximately 30 times compared to that of gold colloids. Potential advantages of using the concept investigated are (1) provision of a rapid and simple immunoassay, (2) satisfaction of a clinical need for highly sensitive determination of analyte, and (3) utilization of relatively inexpensive, portable quantitation means.     

Addressable Microfluidic Polymer Chip for DNA‐Directed Immobilization of Oligonucleotide‐Tagged Compounds

A microfluidic polymer chip for the self‐assembly of DNA conjugates through DNA‐directed immobilization is developed. The chip is fabricated from two parts, one of which contains a microfluidic channel produced from poly(dimethylsiloxane) (PDMS) by replica‐casting technique using a mold prepared by photolithographic techniques. The microfluidic part is sealed by covalent bonding with a chemically activated glass slide containing a DNA oligonucleotide microarray. The dimension of the PDMS–glass microfluidic chip is equivalent to standard microscope slides (76 × 26 mm²). The DNA microarray surface inside the microfluidic channels is configured through conventional spotting, and the resulting DNA patches can be conveniently addressed with compounds containing complementary DNA tags. To demonstrate the utility of the addressable surface within the microfluidic channel, DNA‐directed immobilization (DDI) of DNA‐modified gold nanoparticles (AuNPs) and DNA‐conjugates of the enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP) are carried out. DDI of AuNPs is used to demonstrate site selectivity and reversibility of the surface‐modification process. In the case of the DNA–enzyme conjugates, the patterned assembly of the two enzymes allows the establishment and investigation of the coupled reaction of GOx and HRP, with particular emphasis on surface coverage and lateral flow rates. The results demonstrate that this addressable chip is well suited for the generation of fluidically coupled multi‐enzyme microreactors.     

Negative enrichment of target cells by microfluidic affinity chromatography

A three-dimensional microfluidic channel was developed for high-purity cell separations. This system featured high capture affinity using multiple vertical inlets to an affinity surface. In cell separations, positive selection (capture of the target cell) is usually employed. Negative enrichment, the capture of nontarget cells and elution of target cells, has distinct advantages over positive selection. In negative enrichment, target cells are not labeled and are not subjected to strenuous elution conditions or dilution. As a result, negative enrichment systems are amenable to multistep processes in microfluidic systems. In previous work (Li, P.; Tian, Y.; Pappas, D. Anal. Chem.2011, 83, 774-781), we reported cell capture enhancement effects at vertical inlets to the affinity surface. In this study, we designed a chip that has multiple vertical and horizontal channels, forming a three-dimensional separation system. Enrichment of target cells showed separation purities of 92-96%, compared with straight-channel systems (77% purity). A parallelized chip was also developed for increased sample throughput. A two-channel system showed similar separation purity with twice the sample flow rate. This microfluidic system, featuring high separation purity and ease of fabrication and use is suitable for cell separations when subsequent analysis of target cells is required.     

HRP immobilized microporous Poly(styrene-divinylbenzene-polyglutaraldehyde) monolith for forced flow injected phenol biosensing

A colorimetric detection system based on horseradish peroxidase (HRP) immobilized microporous Poly(styrene-divinylbenzene-polyglutaraldehyde) monolith was developed for phenol biosensing. The incorporation of the aldehyde groups to the backbone of the microporous (90%) monolith was achieved by the polymerization of the continuous phase of a high internal phase emulsion. HRP was chemically immobilized onto the inner walls of the micropores of the monolith via the aldehyde groups. HRP immobilized microporous monolith (HRP-MM) was used as a biocatalytic cell under forced flow conditions in a flow injection system. A carrier stream containing 4-aminoantipyrine (4-AAP) as color reagent was flowed through the HRP-MM at various flow rates ranged between 0.1-15 mL min^-1. The analytical parameters of response time, detection limit and linear range varied at the range of 1-60 s, 1-250 μM, and 0.25-2 mM, respectively. The results showed that the most accurate phenol biosensing was obtained at the flow rate of 1 mL min^-1. The microarchitected structure of the monolith improved the mass transferring conditions for the enzymatic reaction due to the forced flow through the micropores. The detection system presented a satisfactory precision evaluated by relative standard deviation of 1.1% (n = 13) and recovery of 100% at the flow rate of 1 mL min^-1. The system showed 60% of its initial signal from 12th day up to the two months.     

Magneto immunoassays for Plasmodium falciparum histidine-rich protein 2 related to malaria based on magnetic nanoparticles

Magneto immunoassay-based strategies for the detection of Plasmodium falciparum histidine-rich protein 2 (HRP2) related to malaria are described for the first time by using magnetic micro- and nanoparticles. The covalent immobilization of a commercial monoclonal antibody toward the HRP2 protein in magnetic beads and nanoparticles was evaluated and compared. The immunological reaction for the protein HRP2 was successfully performed in a sandwich assay on magnetic micro- and nanoparticles by using a second monoclonal antibody labeled with the enzyme, horseradish peroxidase (HRP). Then, the modified magnetic particles were easily captured by a magneto sensor made of graphite-epoxy composite (m-GEC) which was also used as the transducer for the electrochemical detection. The performance of the immunoassay-based strategy with the electrochemical magneto immunosensors was successfully evaluated and compared with a novel magneto-ELISA based on optical detection using spiked serum samples. Improved sensitivity was obtained when using 300 nm magnetic nanoparticles in both cases. The electrochemical magneto immunosensor coupled with magnetic nanoparticles have shown better analytical performance in terms of limit of detection (0.36 ng mL(-1)), which is much lower than the LOD reported by other methods. Moreover, at a low level of HRP2 concentration of 31.0 ng mL(-1), a signal of 15.30 μA was reached with a cutoff value of 0.34 μA, giving a clear positive result with a non-specific adsorption ratio of 51. Due to the high sensitivity, this novel strategy offers great promise for rapid, simple, cost-effective, and on-site detection of falciparum malaria disease in patients, but also to screen out at-risk blood samples for prevention of transfusion-transmitted malaria.     

Microchip atmospheric pressure chemical ionization source for mass spectrometry

A novel microchip heated nebulizer for atmospheric pressure chemical ionization mass spectrometry is presented. Anisotropic wet etching is used to fabricate the flow channels, inlet, and nozzle on a silicon wafer. An integrated heater of aluminum is sputtered on a glass wafer. The two wafers are jointed by anodic bonding, creating a two-dimensional version of an APCI source with a sample channel in the middle and gas channels symmetrically on both sides. The ionization is initiated with an external corona-discharge needle positioned 2 mm in front of the microchip heated nebulizer. The microchip APCI source provides flow rates down to 50 nL/min, stable long-term analysis with chip lifetime of weeks, good quantitative repeatability (RSD < 10%) and linearity (r(2) > 0.995) with linear dynamic rage of at least 4 orders of magnitude, and cost-efficient manufacturing. The limit of detection (LOD) for acridine measured with microchip APCI at flow rate of 6.2 muL/min was 5 nM, corresponding to a mass flow of 0.52 fmol/s. The LOD with commercial macro-APCI at a flow rate of 1 mL/min for acridine was the same, 5 nM, corresponding to a significantly worse mass flow sensitivity (83 fmol/s) than measured with microchip APCI. The advantages of microchip APCI makes it a very attractive new microfluidic detector.     

Poly(dimethylsiloxane)-based microchip for two-dimensional solid-phase extraction-capillary electrophoresis with an integrated electrospray emitter tip

A microchip in poly(dimethylsiloxane) (PDMS) for in-line solid-phase extraction-capillary electrophoresis-electrospray ionization-time-of-flight mass spectrometry (SPE-CE-ESI-TOF-MS) has been developed and evaluated. The chip was fabricated in a novel one-step procedure where mixed PDMS was cast over steel wires in a mold. The removed wires defined 50-microm cylindrical channels. Fused-silica capillaries were inserted into the structure in a tight fit connection. The inner walls of the inserted fused-silica capillaries and the PDMS microchip channels were modified with a positively charged polymer, PolyE-323. The chip was fabricated in a two-level cross design. The channel at the lower level was packed with 5-microm hyper-cross-linked polystyrene beads acting as a SPE medium used for desalting. The upper level channel acted as a CE channel and ended in an integrated emitter tip coated with conducting graphite powder to facilitate the electrical contact for sheathless ESI. An overpressure continuously provided fresh CE electrolyte independently of the flows in the different levels. Further studies were carried out in order to investigate the electrophoretic and flow rate properties of the chip. Finally, six-peptide mixtures, in different concentrations, dissolved in physiological salt solution was injected, desalted, separated, and sprayed into the mass spectrometer for analysis with a limit of detection in femtomole levels.     

Disposable electrochemical immunosensor diagnosis device based on nanoparticle probe and immunochromatographic strip

We describe a disposable electrochemical immunosensor diagnosis device that integrates the immunochromatographic strip technique with an electrochemical immunoassay and exploits quantum dot (QD, CdS@ZnS) as labels for amplifying signal output. The device takes advantage of the speed and low cost of the conventional immunochromatographic strip test and the high sensitivity of the nanoparticle-based electrochemical immunoassay. A sandwich immunoreaction was performed on the immunochromatographic strip, and the captured QD labels in the test zone were determined by highly sensitive stripping voltammetric measurement of the dissolved metallic component (cadmium) with a disposable screen-printed electrode, which is embedded underneath the membrane on the test zone. The new device coupled with a portable electrochemical analyzer shows great promise for in-field and point-of-care quantitative testing of disease-related protein biomarkers. The parameters (e.g., voltammetric measurement of QD labels, antibody immobilization, the loading amount of QD-antibody, and the immunoreaction time) that govern the sensitivity and reproducibility of the device were optimized with IgG model analyte. The voltammetric response of the optimized device is highly linear over the range of 0.1-10 ng mL(-1) IgG, and the limit of detection is estimated to be 30 pg mL(-1) in association with a 7-min immunoreaction time. The detection limit was improved to 10 pg mL(-1) using a 20-min immunoreaction time. The device has been successfully applied for the detection of prostate-specific antigen (PSA) in human serum sample with a detection limit of 20 pg mL(-1). The results were validated by using the commercial PSA enzyme-linked immunosorbent assay kit and showed high consistency. The new disposable electrochemical diagnosis device thus provides a rapid, clinically accurate, and quantitative tool for protein biomarker detection.     

Microfabricated channel array electrophoresis for characterization and screening of enzymes using RGS-G protein interactions as a model system

A microfluidic chip consisting of parallel channels designed for rapid electrophoretic enzyme assays was developed. Radial arrangement of channels and a common waste channel allowed chips with 16 and 36 electrophoresis units to be fabricated on a 7.62 x 7.62 cm(2) glass substrate. Fluorescence detection was achieved using a Xe arc lamp source and commercial charge-coupled device (CCD) camera to image migrating analyte zones in individual channels. Chip performance was evaluated by performing electrophoretic assays for G protein GTPase activity on chip using BODIPY-GTP as enzyme substrate. A 16-channel design proved to be useful in extracting kinetic information by allowing serial electrophoretic assays from 16 different enzyme reaction mixtures at 20 s intervals in parallel. This system was used to rapidly determine enzyme concentrations, optimal enzymatic reaction conditions, and Michaelis-Menten constants. A chip with 36 channels was used for screening for modulators of the G protein-RGS protein interaction by assaying the amount of product formed in enzyme reaction mixtures that contained test compounds. Thirty-six electrophoretic assays were performed in 30 s suggesting the potential throughput up to 4320 assays/h with appropriate sample handling procedures. Both designs showed excellent reproducibility of peak migration time and peak area. Relative standard deviations of normalized peak area of enzymatic product BODIPY-GDP were 5% and 11%, respectively, in the 16- and 36-channel designs.     

Functional membrane-implanted lab-on-a-chip for analysis of percent HDL cholesterol

A functional lab-on-a-chip has been developed for simultaneous quantitative analyses of high-density lipoprotein (HDL) cholesterol (HDL-C) and total cholesterol (total-C) in a submicroliter plasma sample. The analytical device was fabricated by placing commercial membranes, traditionally used for rapid diagnostics, within microfluidic channels engraved on the surface of a plastic chip. The concentration of HDL-C was measured using enzymatic reactions to produce a colorimetric signal after separation of the single plasma lipoprotein from a mixture. Two small pieces of different membrane pads were used to provide each group of reagents, for HDL separation and enzyme reactions, deposited within their tiny pores in a dry state. To maintain a connection toward the capillary action of the medium, the pads were arranged in a sequence within the fluidic channel that controlled the inlet and outlet of the flow. Upon the addition of a sample, the fluid was delivered through the pads of the chip and a color signal was subsequently generated in proportion to the concentration of HDL-C. The level of total-C was concurrently determined by following identical processes, except absent HDL separation. The two signals were simultaneously determined by employing optical detectors based on transmittance of a light. Such total analyses were completed within 2 min, and the sample sizes were able to be reduced to 0.4 microL for HDL-C and 0.1 microL for total-C, enough to cover the clinically required dynamic ranges.     

Demand information sharing and channel choice in a dual-channel supply chain with multiple retailers

In this paper, under a dual-channel supply chain consisting of a manufacturer and multiple retailers, we investigate vertical and horizontal information sharing in different channel structures and the manufacturer’s choice on whether or not to keep a direct channel. To this end, we first study the dual-channel structure where uncertain demand is a linear function of price with a generalised-distribution base demand and show that the retailers have incentives to share information horizontally but not vertically, while the manufacturer is better off with vertical information sharing but its expected profit is not affected by horizontal information sharing. We next examine the retail-channel structure and find the basic results remain unchanged. Finally, we provide closed-form internal and external conditions under which the manufacturer can benefit from owning a dual-channel structure. Our study extends the existing literature by combining information sharing and dual-channel choice, introducing channel difference, discussing the impact of channel structure on horizontal and vertical sharing as well as providing interesting managerial insights for channel choice.     

Response to cardiac markers in human serum analyzed by guided-mode resonance biosensor

Cardiac markers in human serum with concentrations less than 0.1 ng/mL were analyzed by use of a guided-mode resonance (GMR) biosensor. Cardiac troponin I (cTnI), creatine kinase MB (CK-MB), and myoglobin (MYO) were monitored in the serum of both patients and healthy controls. Dose-response curves ranging from 0.05 to 10 ng/mL for cTnI, from 0.1 to 10 ng/mL for CK-MB, and from 0.03 to 1.7 μg/mL for MYO were obtained. The limits of detection (LOD) for cTnI, CK-MB, and MYO were less than 0.05, 0.1, and 35 ng/mL, respectively. Analysis time was 30 min, which is short enough to meet clinical requirements. Antibody immobilization and the hydrophilic properties of the guided-mode resonance filter (GMRF) surface were investigated by X-ray photoelectron spectroscopy (XPS) and by monitoring the peak wavelength shift and water contact angle (CA). Both assays used to evaluate the surface density of the immobilized antibodies, a sandwich enzyme-linked immunosorbent assay (ELISA) and a sandwich immunogold assay, showed that the antibodies were successfully immobilized and sufficiently aligned to detect the low concentration of biomarkers. Our results show that the GMR biosensor will be very useful in developing low-cost portable biosensors that can screen for cardiac diseases.     

Electrophoretically mediated microanalysis of leucine aminopeptidase using two-photon excited fluorescence detection on a microchip

Two-photon excited fluorescence detection was performed on a microfabricated electrophoresis chip. A calibration curve of the fluorescent tag beta-naphthylamine was performed, resulting in a sensitivity of 2.5 x 10(9) counts M(-1) corresponding to a detection limit of 60 nM. Additionally, leucine aminopeptidase was assayed on the chip using electrophoretically mediated microanalysis. The differential electroosmotic mobilities of the enzyme and substrate, L-leucine beta-naphthylamide, allowed for efficient mixing in an open channel, resulting in the detection of a 30 nM enzyme solution under constant potential. A zero potential incubation for 1 min yielded a calculated detection limit of 4 nM enzyme.     

Competitive stocking and coordination in a multiple-channel distribution system

In this paper, we study a multiple-channel distribution system in which a manufacturer sells its product through an independent retailer as well as through his wholly-owned channel. The manufacturer and the retailer stock the product solely to satisfy the final customer demand of their respective channels. We focus on the stocking levels of the manufacturer's wholly-owned channel and the retail channel. We assume that each channel has a local stochastic demand, but that the products are substitutable, which means there will be spill-over customers in the event that one channel runs out of stock. We explore the channel inefficiencies induced by the presence of simultaneous vertical competition (double-marginalization) and horizontal competition (substitutability). We show that there is an overall tendency for both channels to overstock due to substitution, which intensifies under increasing substitution rates. Increasing double-marginalization, on the other hand, intensifies the tendency to overstock in the manufacturer's wholly-owned channel, but induces the retail channel to understock. We find that supply chain losses are least under moderate levels of double-marginalization and or substitution. We also investigate coordination mechanisms, and show that most of the well-known, simple contracts fail to achieve coordination in this setting. An exception to this is an appropriately designed penalty contract which can indeed coordinate the supply chain, but is hard to implement. In search of practically more appealing coordination mechanisms, we design a novel two-part compensation-commission contract, whose terms depend on the retail channel sales.     

MEDUSA-32: A low noise, low power silicon strip detector front-end electronics, for space applications

In this report we describe a mixed analog-digital integrated circuit (IC) designed as the front-end electronics for silicon strip-detectors for space applications. In space power consumption, compactness and robustness become critical constraints for a pre-amplifier design. The IC is a prototype with 32 complete channels, and it is intended for a large area particle tracker of a new generation of gamma ray telescopes. Each channel contains a charge sensitive amplifier, a pulse shaper, a discriminator and two digital buffers. The reference trip point of the discriminator is adjustable. This chip also has a custom PMOSFET transistor per channel, included in order to provide the high dynamic resistance needed to reverse-bias the strip diode.

The digital part of the chip is used to store and serially shift out the state of the channels. There is also a storage buffer that allows the disabling of non-functioning channels if it is required by the data acquisition system.

An input capacitance of 30 pF introduced at the input of the front-end produces less than 1000 electrons of RMS equivalent noise charge (ENC), for a total power dissipation of only 60 μW per channel.

The chip was made using Orbit's 1.2 μm double poly, double metal n-well low noise CMOS process. The dimensions of the IC are 2400 μm × 8840 μm.     

Isoelectric focusing in a microfluidically defined electrophoresis channel

A new form of microchip isoelectric focusing that allows efficient coupling with pretreatment processes is reported. The sample is conveyed in a carrier ampholyte solution to the separation channel that is connected at both ends by two V-shaped lead channels, which supply electrode solutions to the connection point and complete the electrical connection to off-chip electrodes. The relatively high electric conductivity of the electrode solutions compared with that of the pH gradient enables focusing with a 2% loss of applied voltage at the electrodes using the lead channels. A glass microchip was constructed specifically for this configuration. The channel wall was coated with polydimethylacrylamide, and the IEF chip was operated in a chip holder equipped with on-chip connector valves. A plug of fluorescence-labeled peptide p I markers with p I values ranging from 3.64 to 9.56 with carrier ampholyte solution (pH 3-10) was introduced into the separation channel. When the plug reached the channel segment (24 mm in length) between the connection points with the electrolyte lead channels, isoelectric focusing was started after filling the lead channels with electrolyte solutions. The peptide markers were observed using scanning fluorescence detection. The entire range of the pH gradient was established in the segment after approximately 2 min. Isoelectric focusing of three consecutively injected sample plugs containing different p I markers was demonstrated.     

Perfusion in microfluidic cross-flow: separation of white blood cells from whole blood and exchange of medium in a continuous flow

We describe a microfluidic technique for separation of particles and cells and a device that employs this technique to separate white blood cells (WBC) from whole human blood. The separation is performed in cross-flow in an array of microchannels with a deep main channel and large number of orthogonal, shallow side channels. As a suspension of particles advances through the main channel, a perfusion flow through the side channels gradually exchanges the medium of the suspension and washes away particles that are sufficiently small to enter the shallow side channels. The microfluidic device is tested with a suspension of polystyrene beads and is shown to efficaciously exchange the carrier medium while retaining all beads. In tests with whole human blood, the device is shown to reduce the content of red blood cells (RBC) by a factor of approximately 4000 with retention of 98% of WBCs. The ratio between WBCs and RBCs reached at an outlet of the device is 2.4 on average. The device is made of a single cast of poly(dimethylsiloxane) sealed with a cover glass and is simple to fabricate. The proposed technique of separation by perfusion in continuous cross-flow could be used to enrich rare populations of cells based on differences in size, shape, and deformability.     

Microfabricated renewable beads-trapping/releasing flow cell for rapid antigen-antibody reaction in chemiluminescent immunoassay

A renewable flow cell integrating a microstructured pillar-array filter and a pneumatic microvalve was microfabricated to trap and release beads. A bead-based immunoassay using this device was also developed. This microfabricated device consists of a microfluidic channel connecting to a beads chamber in which the pillar-array filter is built. Underneath the filter, there is a pneumatic microvalve built across the chamber. Such a device can trap and release beads in the chamber by "closing" or "opening" the microvalve. On the basis of the pneumatic microvalve, the device can trap beads in the chamber before performing an assay and release the used beads after the assay. Therefore, this microfabricated device is suitable for "renewable surface analysis". A model analyte, 3,5,6-trichloropyridinol (TCP), was chosen to demonstrate the analytical performance of the device. The entire fluidic assay process, including beads trapping, immuno binding, beads washing, beads releasing, and chemiluminesence signal collection, could be completed in 10 min. The immunoassay of TCP using this microfabricated device showed a linear range of 0.20-70 ng/mL with a limit of detection of 0.080 ng/mL. The device was successfully used to detect TCP spiked in human plasma at the concentration range of 1.0-50 ng/mL, with an analytical recovery of 81-110%. The results demonstrated that this device can provide a rapid, sensitive, reusable, low-cost, and automatic tool for detecting various biomarkers in biological fluids.     

A numerical study on the heat-transfer characteristics of an array of alternating horizontal or vertical oval cross-section pipes placed in a cross stream

In this paper, a numerical study on the flow of an array of alternating horizontal or vertical oval cross-section pipes placed in a cross stream, resembling that encountered in a real cross-flow heat exchanger, is presented. The results include temperature contours and velocity vectors on the surface of the pipe and at several selected cross-sections, the local and sectional averaged Nusselt number distributions, and the overall Nusselt number variations versus the internal and external flow Reynolds numbers. The computation shows that the wall temperature varies dramatically along both the circumferential and the axial directions of the pipe, and is far different from a constant wall temperature distribution assumed in most previous studies. In general, the portion of the pipe with external wall facing forwards the cross-flow stream has lower temperature and higher local heat-transfer rate than the portion with wall facing backwards the cross-flow stream. At the maximum external flow Reynolds number, Re = 40, considered in this paper, the magnitude of pipe's overall Nusselt number is about one-sixth the magnitude of that calculated based on the ideal constant wall temperature assumption.     

层间微通道液体冷却3D-ICs的仿真研究

近年来,随着半导体工业的迅速发展,芯片特征尺寸逐渐减小逐渐接近极限,为此提出三维集成电路(3D-ICs),集成度显著提高,但同时也造成芯片功率密度成倍增加,层间微通道液体冷却因其结构紧凑、传热效果较好、压降低等优点成为备受关注的焦点。本文采用仿真工具3D-ICE建立带有层间微通道液体冷却的不同通道类型的3D-ICs模型,模拟分析层间通道的物性参数如通道壁厚/针肋直径、通道高度、制冷剂流速/达西速度对三维芯片温度分布的影响情况。结果表明,给定条件下,热点温度随通道壁厚/针肋直径的增加而减少,在50~100变化快,温降最高可达1.309℃,随后趋于稳定;热点温度随通道高度变化的变化因通道类型而异,矩形直通道Tmax在0~1间迅速降低,随后逐渐升高,线性微针肋Tmax在一定范围内较矩形直通道平缓下降,随后缓慢升高或趋于平稳;热点温度随制冷剂流速/达西速度的增加而降低,且变化逐渐平缓。