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Abstract
We present a flexible, polymer based MEMS differential scanning calorimetric (DSC) device combining integrated microfluidic channels, highly sensitive thermoelectric sensing, and real-time temperature monitoring for thermodynamic characterization of biomolecular samples with minimized sample consumption. The device uses an inexpensive, commercially available polymer substrate and a novel fabrication approach to create a microstructure consisting of a pair of microchannels (containing the sample and reference buffer, respectively), which are integrated with resistive temperature sensors (for in-situ measurement of sample temperature) and an antimony-bismuth (Sb-Bi) thermopile (for measurement of the temperature difference between the sample and reference channels). We demonstrate the utility of this MEMS DSC device by measuring the unfolding of lysozyme in a small volume (1 μL), and at practically relevant protein concentrations (approaching 1 mg/mL). Thermodynamic properties including the total enthalpy change per mole of protein (ΔH) and melting temperature (Tm) at different protein concentrations during this conformational transition are determined and found to agree with published data.
... MEMS technology has been used to fabricate a variety of integrated analytical devices, particularly, calorimetric sensors, due to their excellent thermal isolation property in the microscale as well as significantly reduced thermal mass and sample volume [1][2][3]. The MEMS based DSC offers attractive advantages such as improved sensitivity, reduced power consumption, higher scanning rate, and much smaller device scale [4]. ...
Thin film based differential scanning calorimeter (DSC) was developed and utilized for the study of phase-transition kinetics in protein samples. In this paper, we proposed a MEMS based DSC model. The distance between the sample and reference cell was optimized based on the thermal analysis result. The influence of the response time to the shape of the DSC thermograms and the transition temperature was also studied systematically. Meanwhile, Lumry-Eyring model was used to investigate the relationship between the scanning rate and the transition temperature. The modeling result was verified by the lysozyme denaturation tests. This study provided a pathway for the optimization of MEMS based DSC array and the correction of the DSC thermograms.
This paper presented a power compensated MEMS differential scanning calorimeter (DSC) for protein stability characterization. In this microfabricated sensor, PDMS (Polydimethylsiloxane) and polyimide were used to construct the adiabatic chamber (1 μL) and temperature sensitive vanadium oxide was used as the thermistor material. A power compensation system was implemented to maintain the sample and reference at the same temperature. The resolution study and step response characterization indicated the high sensitivity (6 V/W) and low noise level (60 μk) of the device. The test with IgG1 antibody (mAb1) samples showed clear phase transitions and the data was confirmed to be reasonable by comparing it with the results of commercial DSC’s test. This device used ∼1uL sample amount and could complete the scanning process in 4 minutes, significantly increasing the throughput of the bimolecular thermodynamics study like drug formulation process.
A facile and convenient approach has been developed for the synthesis of functionalized indazoles via solid state melt reaction using easily accessible starting materials under catalyst-free conditions. This transformation involves electrocyclization via a conjugated nitrene intermediate obtained under thermal conditions. Further anti-tubercular activity screening of the molecules was undertaken, among the compounds 3a-3x screened for in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv, compound 3u (MIC: 4.20 μM) was found to be most active and are superior over existing standard drugs ciprofloxacin and ethambutol. Compounds 3c and 3x were found to equally potent as ethambutol. Among most potent compounds in the series, four compounds (3n, 3o, 3p and 3u) showed lower cytotoxicity which could be promising drug candidates for further development.
MEMS based micro heaters are a key component in micro bio-calorimetry, nondispersive infrared gas sensors, semiconductor gas sensors and microfluidic actuators. A micro heater with a uniform temperature distribution in the heating area and short response time is desirable in ultrasensitive temperature-dependent measurements. In this paper we propose a novel micro heater design to reach a uniform temperature in a large heating area by optimizing the heating power density distribution in the heating area. A polyimide membrane is utilized as the substrate to reduce the thermal mass and heat loss which allows for fast thermal response as well as a simplified fabrication process. A gold and titanium heating element is fabricated on the flexible polyimide substrate using the standard MEMS technique. The temperature distribution in the heating area for a certain power input is measured by an IR camera, and is consistent with FEA simulation results. This design can achieve fast response and uniform temperature distribution, which is quite suitable for the programmable heating such as impulse and step driving.
We developed an ultrasensitive micro-DSC (differential scanning calorimeter) for liquid protein sample characterization. This design integrated vanadium oxide thermistors and flexible polymer substrates with microfluidics chambers to achieve a high sensitivity (6 V/W), low thermal conductivity (0.7 mW/K), high power resolutions (40 nW), and well-defined liquid volume (1 μl) calorimeter sensor in a compact and cost-effective way. We further demonstrated the performance of the sensor with lysozyme unfolding. The measured transition temperature and enthalpy change were in accordance with the previous literature data. This micro-DSC could potentially raise the prospect of high-throughput biochemical measurement by parallel operation with miniaturized sample consumption.
Isothermal titration calorimetry (ITC) directly measures heat evolved in a chemical reaction to determine equilibrium binding properties of biomolecular systems. Conventional ITC instruments are expensive, use complicated design and construction, and require long analysis times. Microfabricated calorimetric devices are promising, although they have yet to allow accurate, quantitative ITC measurements of biochemical reactions. This paper presents a microfabrication-based approach to integrated, quantitative ITC characterization of biomolecular interactions. The approach integrates microfabricated differential calorimetric sensors with microfluidic titration. Biomolecules and reagents are introduced at each of a series of molar ratios, mixed, and allowed to react. The reaction thermal power is differentially measured, and used to determine the thermodynamic profile of the biomolecular interactions. Implemented in a microdevice featuring thermally isolated, well-defined reaction volumes with minimized fluid evaporation as well as highly sensitive thermoelectric sensing, the approach enables accurate and quantitative ITC measurements of protein-ligand interactions under different isothermal conditions. Using the approach, we demonstrate ITC characterization of the binding of 18-Crown-6 with barium chloride, and the binding of ribonuclease A with cytidine 2'-monophosphate within reaction volumes of approximately 0.7µL and at concentrations down to 2mM. For each binding system, the ITC measurements were completed with considerably reduced analysis times and material consumption, and yielded a complete thermodynamic profile of the molecular interaction in agreement with published data. This demonstrates the potential usefulness of our approach for biomolecular characterization in biomedical applications.
We have developed a bulk micromachined calorimeter with a sensitivity of 1.5 nW / Hz 1/2 and a 1 ms time constant using a thin film thermopile as the sensing element. The thermopile consists of seven titanium and bismuth thermocouples with a total Seebeck coefficient of 574 μ V / K . The device is capable of measuring enthalpies in chemical or biological reactions in volumes as small as a few picoliters. The device can be fabricated and operated in a massively parallel fashion in combination with ink-jet printing technologies in air and at room temperature, making it ideally suited for biological and biochemical experiments.
We report the fabrication of enthalpy arrays and their use to detect molecular interactions, including protein-ligand binding, enzymatic turnover, and mitochondrial respiration. Enthalpy arrays provide a universal assay methodology with no need for specific assay development such as fluorescent labeling or immobilization of reagents, which can adversely affect the interaction. Microscale technology enables the fabrication of 96-detector enthalpy arrays on large substrates. The reduction in scale results in large decreases in both the sample quantity and the measurement time compared with conventional microcalorimetry. We demonstrate the utility of the enthalpy arrays by showing measurements for two protein-ligand binding interactions (RNase A + cytidine 2'-monophosphate and streptavidin + biotin), phosphorylation of glucose by hexokinase, and respiration of mitochondria in the presence of 2,4-dinitrophenol uncoupler.
Calorimetry offers a direct measurement of thermodynamic properties of materials, including information on the energetics of phase transitions. Many materials can only be prepared in thin film or small crystal (submilligram) form, negating the use of traditional bulk techniques. The use of micromachined, membrane-based calorimeters for submilligram bulk samples is detailed here. Numerical simulations of the heat flow for this use have been performed. These simulations describe the limits to which this calorimetric technique can be applied to the realm of small crystals (1-1000 microg). Experimental results confirm the feasibility of this application over a temperature range from 2 to 300 K. Limits on sample thermal conductivity as it relates to the application of the lumped and distributed tau 2 models are explored. For a typical sample size, the simulations yield 2.5% absolute accuracy for the heat capacity of a sample with thermal conductivity as low as 2 x 10(-5) W/cm K at 20 K, assuming a strong thermal link to the device. Silver paint is used to attach (both thermally and physically) the small samples; its heat capacity and reproducibility are discussed. Measurements taken of a submilligram single crystal of cobalt oxide (CoO) compare favorably to the results of a bulk calorimetric technique on a larger sample.
For the new Flash DSC 1, the temperature windows-to-operate—the temperature ranges where the real, achieved scan rate is constant—have been determined for unloaded sensors under various conditions like purge gas and flow rate variations; cooling to −90 °C and heating to 450 °C; scan rates from 1 up to 20,000 °C s−1 in heating and 15,000 °C s−1 in cooling. Compared to nitrogen, helium purge gas offers better access to low-temperature transitions and enables faster cooling. Drawback is the decreased temperature window-to-operate in heating at the high-temperature side. The temperature calibration protocol according to the recent DIN SPEC 91127 for sample mass and scan rate was found to be useful. The correction factors are maximal −1.4 °C as measured for 1 μg at 1,000 °C s−1 heating. Using liquid crystalline substances it was proved that the Flash DSC 1 has symmetry, meaning that calibration data found in heating also can be applied in cooling.
Graphical abstract
The heat capacity, Cp, of glycerol was measured between 298 and 383K with a DSC and a modulated scanning calorimeter operating at 5mHz. The two sets of data show an excellent agreement and can be fitted to the equation: Cp (Jmol−1K−1)=90.983+0.4335T. The Cp values, at 298.15K, tabulated in literature and in data handbooks agree with the current data, but the equation reported for Cp(T) at higher temperatures is wrong, giving a slope which is ca. 60% of the determined value.
The differential scanning calorimeter (d.s.c.) has been widely used to determine the thermodynamics of phase transitions and conformational changes in biological systems including proteins, nucleic acid sequences, and lipid assemblies. The d.s.c monitors the temperature difference between two vessels, one containing the biological solution and the other containing a reference solution, as a function of temperature at a given scan rate. Recommendations for d.s.c. measurement procedures, calibration procedures, and procedures for testing the performance of the d.s.c. are described. Analysis of the measurements should include a correction for the time response of the instrument and conversion of the power versus time curve to a heat capacity versus temperature plot. Thermodynamic transition models should only be applied to the analysis of the heat capacity curves if the model-derived transition temperatures and enthalpies are independent of the d.s.c. scan rate. Otherwise, kinetic models should be applied to the analysis of the data. Application of thermodynamic transition models involving two states, two states and dissociation, and three states to the heat capacity versus temperature data are described. To check the operating performance with standard d.s.c.s, samples of (1 to 10) mg · cm3solutions of hen egg white lysozyme in 0.1 mol · dm−3HCl–glycine buffer at pH = (2.4 ± 0.1) were sent to six different d.s.c. laboratories worldwide. The values obtained from proper measurements and application of a two-state transition model yielded an average unfolding transition temperature for lysozyme of 331.2 K with values ranging between T= 329.4 K and 331.9 K, and an average transition enthalpy of 405 kJ · mol−1with values ranging from 377 kJ · mol−1to 439 kJ · mol−1. It is recommended that the reporting of d.s.c. results be specific with regard to the composition of the solution, the operating conditions and calibrations of the d.s.c. determination of base lines that may be model dependent, and the model used in the analysis of the data.
In an effort to supply accurate heat capacity data on pure materials which will serve as standards in precision calorimetry, there are given the results of new heat capacity measurements at the National Bureau of Standards on n-heptane (25° to 523°K.) and on aluminum oxide (corundum), (14 to 1173°K.). These materials together with benzoic acid had been recommended by the Calorimetry Conference (U. S.) as standards for the intercomparison of calorimeters. Diphenyl ether is suggested as an additional standard to supplement the above three substances and water which is universally used as a standard. Tables of smoothed values of heat capacity and enthalpy are given for n-heptane, aluminum oxide, benzoic acid (up to 430° K.), diphenyl ether (up to 570°K.) and water (0 to 100°C.).
Modulated DSCTM (MDSC) is a new, patent-pending extension to conventional DSC which provides information about the reversing and nonreversing
characteristics of thermal events, as well as the ability to directly measure heat capacity. This additional information aids
interpretation and allows unique insights into the structure and behaviour of materials., A number of examples of its use
are described.
Modulierte DSCTM (MDSC) ist eine angemeldete Patenterweiterung herkömmlicher DSC, welche Informationen über reversible und nichtreversible
Eigenschaften von thermischen Geschehnissen erstellt und sich zur direkten Messung der Wärmekapazität eignet. Diese zusätzlichen
Informationen helfen bei der Interpretation und erlauben ein eindeutiges Verständnis des Verhaltens von Substanzen. Es wird
eine Anzahl von Anwendungsbeispielen diskutiert.
The metabolism of cells is studied by scientists using a number of cells high enough to have a signal in the regular differential
scanning calorimetry (DSC). Currently the number of cells necessary for thermal analysis is on the order of hundreds of thousands.
The cell metabolism researchers have been trying to reduce this number with an eye on the final prize of sample size of one.
In this paper, we report a novel MEMS_DSC (Micro Electro Mechanical System-Differential Scanning Calorimetry), which reduces
the number of cells needed for analysis by about two orders of magnitude. It uses the Seebeck effect generated by a polysilicon
n-gold junction to convert the temperature measured from cells directly into a voltage signal. To characterize the device
a laser was used to heat the sensor; a lock-in amplifier, coupled with an optical chopper, allowed us to measure a very low
heat power (25.5nW).
Thick polyimide layers can be formed by using some unique properties of poly(dimethylsiloxane)-polyimide (PDMS/PMDA–ODA) blends followed by surface modification and deposition of a second layer of polyimide precursor chemicals. The method is based on the micro-phase separation characteristics of these blends to yield surfaces that have PDMS-like character. Upon modification with UV/ozone treatment, a surface that is essentially SiOx
and hydrophilic in nature is produced. This surface is amenable to reaction and deposition of a second polyimide layer from polyimide precursors. The thicker polyimide layer has enhanced adhesion between the original layer of the blend and the new polyimide layer and this approach finds extensive applications for products that require thick polymer layers. Changes in surface energy for various blend compositions were monitored by measurement of advancing contact angle with de-ionized water. Contact angle for unmodified polyimide films was on the order of 70° and it increased to about 104° after blending with PDMS and curing. UV/ozone treatment reduced the contact angle of the doped polyimide to less than 5°. X-ray photoelectron spectroscopy (XPS) and angle resolved XPS (ARXPS) measurements were used to monitor the chemical compositions of the various surfaces. High-resolution XPS spectra in the Si2p region confirm the transformation of O–Si–C bonds in PDMS to SiOx
, where x is about 2. Scanning electron microscopy (SEM) of some selected samples shows that the blends contain phase separation of the polymers at the surfaces of the samples. Atomic force microscopy (AFM) of siloxane-free polyimide, and PDMS/PMDA–ODA blends both prior to and after UV/ozone exposure, show that the films are essentially flat at short treatment times (less than 60 min). AFM also reveals the separation of PDMS into micro-domains at the cured film surface and throughout the layer below the surface of the blended films. Adhesion of a subsequently deposited polyimide layer to the modified polyimide surface was found to be greatly improved when compared to the adhesion obtained for deposition onto a pristine polyimide surface.
We present a MEMS-based differential scanning calorimetric (DSC) device combining highly sensitive thermoelectric sensing, on-chip self-calibration, and microfluidic regulation for thermodynamic characterization of biomolecular samples on a minimized scale. The device integrates well-defined microfluidic reaction chambers and utilizes a three-dimensional structure in which a layer of resistive microheaters and temperature sensors are precisely aligned to these chambers to provide uniform heating, in-situ temperature sensing, and convenient self-calibration. Notably, this device exploits the novel use of an antimony-bismuth (Sb-Bi) thermopile with high thermoelectric performance to significantly enhance device sensitivity and thus allow for DSC detection with minimized sample consumption. We demonstrate the utility of this MEMS DSC device by characterizing the unfolding of proteins in a minimized volume (1 μL), and at low protein concentrations approaching practically useful levels (1 mg/mL). Quantitative thermodynamic properties including the total enthalpy change (ΔH) and melting temperature (Tm) during this conformational transition are determined and found to agree with published data.
The heat capacities of various liquids were determined with a high precision using differential scanning calorimetry. The calibration and measurement procedures, as implemented on a Setaram Micro DSC II calorimeter based on Calvet’s differential detection of the heat flow rate in the measuring and reference vessels are analysed and discussed. An experimental procedure for determining heat capacities based on the scanning method is proposed. Calibration is done with liquids of known heat capacity. The proposed method was checked against various standard liquids. The results of ensuing scanning method are compared with those obtained by using the isothermal step method.
We demonstrate microelectromechanical system (MEMS)-based differential scanning calorimetry (DSC) for characterizing thermodynamic properties of biomolecules. The MEMS device consists of a pair of polydimethylsiloxane (PDMS) calorimetric microchambers and fluid handling microchannels, with each chamber 1.2 μl in volume and based on a freestanding SU-8 diaphragm. A nickel–chromium thermopile, and nickel heaters and temperature sensors, are integrated on the diaphragms to allow thermal measurement and control. During DSC measurements, the chambers are filled with a biomolecular solution and reference buffer, respectively. As the solution temperatures are varied continuously over a range of interest, the biomolecular thermal power is measured via the thermopile output, and then used to compute the thermodynamic parameters of the biomolecule. We present results from applying the device to DSC measurements of the unfolding of the proteins lysozyme and ribonuclease A. The enthalpy of unfolding and melting temperature of the proteins obtained are in agreement with published data.