Measure energy – the fundament of life

calScreener™ is the first of its kind cell-biology optimized isothermal microcalorimeter for bioactivity measurements.

Calorimetry - a new paradigm in cell-based assays

Direct metabolic assays with isothermal calorimetry stand out from reductionistic biochemical assays overcoming the limitations of parameter based analyses with unique characteristics.

Measuring the metabolic activities in living organisms is a well established science. In 1784, Antoine Laurent Lavoisier and Pierre Simon de Laplace cleverly devised the first calorimetric device. Calorimeters have since evolved as modern tools for the advancement of science. Instruments have however not been adapted for biology and drug development. Not until now.

The calScreener is the calorimetry instrument for the biologist by increasing throughput, minimizing sample size yet increasing sensitivity, optimized sterile plastic growth inserts for ease-of-use and user-friendly software with customized analysis.

The calScreener™ principle

Biological processes caused by physical, chemical or biological stimuli in which metabolic changes are anticipated are all valid for the analysis.
The calPlate™ containing the individual sealed cups holding the cell culture are placed in a thermostated chamber set at the target temperature with a precision within thousands of a Kelvin.

The cups rest upon a heat-flux detecting sensor, the thermopile. The sensor is attached to a heat-sink with a large mass compared to the cell-culture cups. All heat produced is transferred to the heat-sink giving rise to a signal in the thermopile sensor proportional to the heat-flow.

The measured heat is thus independent of the model system or the process involved. We have a label free, real-time, detection system applicable to a wide range of biological applications.

Illustration of how the heat sensors work when the calPlate in inserted into the calScreener

The new gold standard of metabolic measurements

The term isothermal microcalorimeter is commonly used for calorimeters designed for experiments in the microwatt range and lower, under isothermal conditions. Practically all such calorimeters are of the heat flow (heat conduction) type using semiconductor thermopiles (thermoelectric plates) as sensors for the heat flow between the calorimetric vessel and a surrounding heat sink. Isothermal microcalorimeters are normally designed as twin instruments to increase sensitivity.

The thermal power, P, (rate of heat production) of living organisms is related to its metabolic rates and P may therefore be taken as a quantitative measure of its “activity”. All metabolically active organisms produce heat, making isothermal calorimetry a general technique for the monitoring of their activities. Measurements of P can be made without any labeling of the sample and the technique can be applied under oxic or anoxic conditions for the cellular systems in suspension, in sediments or attached to solid surfaces. P can be monitored continuously, without any interference with the samples and over long periods of time (days, on the µW level). Furthermore, isothermal microcalorimetric techniques typically have higher reproducibility and detectability than most other methods used in measurements of a functional property of living organisms.

Isothermal microcalorimeters are used in fundamental thermodynamic work and in determination of kinetic properties of slow chemical and biochemical reactions. Isothermal microcalorimetic techniques have also found use as general monitors for different types of complex processes, for example in the estimation of stabilities of technical products like drugs and in determination of activities of living organisms. For more than half a century it has repeatedly been stated that such techniques are of potential practical importance in several areas of applied biology. A large number of methodological studies have been reported, in particular in areas related to medicine and environmental work. Important progress has been made in experimental techniques and essential experimental problems have been identified, but the low rate of sample throughput for heat flow calorimeters has limited their use in practical work. That disadvantage can now be overcome by use of multi-channel instruments, where an assembly of calorimetric units (“channels”) makes it possible to measure many samples in one experiment.

The cell-based assay arena is slowly entering center stage due to the major and growing medical problems facing the world—both financial (i.e., drug development) and curative (i.e., antibiotic resistance). In a modern 3-D holistic format, the mature technique of calorimetry is the answer to some of these problems.

This article describes how and why measurement of the total metabolic response of a biological system is a valuable proposition and a true time- and cost-efficient complement to existing assays.

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Antibiotic-resistant bacteria are a growing concern, and with good reason—an estimated 23,000 deaths and two million illnesses occur annually in the U.S.1 Major contributing factors are overuse, lack of regulation and scarcity of novel antibiotics.

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Isothermal microcalorimetry is a label-free assay that allows monitoring of enzymatic and metabolic activities. The technique has strengths, but most instruments have a low throughput, which has limited their use for bioassays. Here, an isothermal microcalorimeter, equipped with a vessel holder similar to a 48-well plate, was used. The increased throughput of this microcalorimeter makes it valuable for biomedical and pharmaceutical applications.

Our results show that the sensitivity of the instrument allows the detection of 3 × 10(4) bacteria per vial. Growth of P. mirabilis in Luria Broth medium was detected between 2 and 9 h with decreasing inoculum. The culture released 2.1J with a maximum thermal power of 76 μW. The growth rate calculated using calorimetric and spectrophotometric data were 0.60 and 0.57 h(-1) , respectively. Additional insight on protease activities of P. mirabilis matching the last peak in heat production could be gathered as well. Growth of tumor microtissues releasing a maximum thermal power of 2.1 μW was also monitored and corresponds to a diameter increase of the microtissues from ca. 100 to 428 μm. This opens new research avenues in cancer research, diagnostics, and development of new antitumor drugs.

For parasitic worms, the technique allows assessment of parasite survival using motor and metabolic activities even with a single worm.

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Soil-transmitted helminths, which affect the poorest communities, worldwide cause a range of symptoms and morbidity, yet few treatment options are available and drug resistance is a concern.

To improve and accelerate anthelminthic drug discovery, novel drug screening tools such as isothermal microcalorimetry (IMC) have been tested with great potential. In this study, we used a novel microcalorimeter, the calScreener™, to study the viability on the hookworms Necator americanus and Ancylostoma ceylanicum as well as the whipworm Trichuris muris. Significant heat flow signals could be obtained with already one adult worm per channel for all three species. High-amplitude oscillations were observed for the hookworms; however, adult T. muris showed a twofold heat flow decrease during the first 24 h.

Antinematodal effects of ivermectin and levamisole at 1, 10, and 100 μg/ml were evaluated on adult N. americanus and A. ceylanicum. Levamisole-treated hookworms showed a decline in heat flow and oscillation amplitude in a dose-response manner. Heat flow for ivermectin-treated hookworms increased proportionally with increased concentrations of ivermectin, though the wavelet analysis showed an opposite trend as observed by flatter wavelets. In conclusion, the calScreener™ is an excellent tool to study drug effects on intestinal hookworms at the adult worm stage as it offers a lower detection limit than other IMC devices and the possibility to monitor worm viability online.

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Caries-associated biofilms induce loss of calcium from tooth surfaces in the presence of dietary carbohydrates. Exopolysaccharides (EPS) provide a matrix scaffold and an abundance of primary binding sites within biofilms. The role of EPS in binding calcium in cariogenic biofilms is only partially understood. Thus, the aim of the present study is to investigate the relationship between the calcium dissolution rates and calcium tolerance of caries-associated bacteria and yeast as well as to examine the properties of EPS to quantify its binding affinity for dissolved calcium. Calcium dissolution was measured by dissolution zones on Pikovskaya’s agar.

Calcium tolerance was assessed by isothermal microcalorimetry (IMC) by adding CaCl2 to the bacterial cultures. Acid-base titration and Fourier transform infrared (FTIR) spectroscopy were used to identify possible functional groups responsible for calcium binding, which was assessed by isothermal titration calorimetry (ITC). Lactobacillus spp. and mutans streptococci demonstrated calcium dissolution in the presence of different carbohydrates.

All strains that demonstrated high dissolution rates also revealed higher rates of calcium tolerance by IMC. In addition, acidic functional groups were predominantly identified as possible binding sites for calcium ions by acid-base titration and FTIR. Finally, ITC revealed EPS to have a higher binding affinity for calcium compared, for example, to lactic acid. In conclusion, this study illustrates the role of EPS in terms of the calcium tolerance of cariogenic microbiota by determining the ability of EPS to control free calcium concentrations within the biofilms as a self-regulating mode of action in the pathogenesis of dental caries.

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Design and properties are reported for a novel type of multi-channel isothermal microcalorimeter. It is equipped with 48 calorimetric units (channels) and is primarily intended for use as a monitor of the activity of living cells, tissues and small animals. Calorimetric vessels are positioned in a holder with the format of a 48-well microtiter plate.

At most, 47 samples can be measured simultaneously; one vessel is then used as reference. The standard configuration is 32 sample positions using 16 channels as references in a twin calorimeter setup. The detection limit is then 0.1 μW. Sample volumes are usually 100 μl–300 μl. The 24 h baseline stability is typically 0.2 μW (room temperature variation ≤1 °C). The instrument was designed considering feasible uses in applied biology, especially in pharmaceutical and clinical laboratories and in environmental work.

However, it can be employed as a general monitor of slow processes in different fields of biology and for non-biological systems, including accurate determination of their thermal powers (heat production rates). In the present report, properties of the instrument are characterized by chemical calibration experiments and in measurements of growth of bacteria and mammalian cells.

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The identification of brown adipose deposits in adults has led to significant interest in targeting this metabolically active tissue for treatment of obesity and diabetes.

Improved methods for the direct measurement of heat production as the signature function of brown adipocytes (BAs), particularly at the single cell level, would be of substantial benefit to these ongoing efforts. Here, we report the first application of a small molecule-type thermosensitive fluorescent dye, ERthermAC, to monitor thermogenesis in BAs derived from murine brown fat precursors and in human brown fat cells differentiated from human neck brown preadipocytes.

ERthermAC accumulated in the endoplasmic reticulum of BAs and displayed a marked change in fluorescence intensity in response to adrenergic stimulation of cells, which corresponded to temperature change. ERthermAC fluorescence intensity profiles were congruent with mitochondrial depolarisation events visualised by the JC-1 probe. Moreover, the averaged fluorescence intensity changes across a population of cells correlated well with dynamic changes such as thermal power, oxygen consumption, and extracellular acidification rates.

These findings suggest ERthermAC as a promising new tool for studying thermogenic function in brown adipocytes of both murine and human origins.

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