CALIBRATION OF A CHIP-BASED MICROFLUIDIC CALORIMETER

Abstract

The invention provides a calibration method for calibrating a chip-based microfluidic calorimeter, wherein the chip-based microfluidic calorimeter comprises one or more thermopiles, wherein the calibration method uses the deprotonating reaction of a phosphate group, the method comprising: providing calibration liquids comprising (i) a buffer with a pH range of at least 7-9 and (ii) a first compound with a phosphate group which is protonated in a pH range of at least 3-6, and mixing these calibration liquids in the chip-based microfluidic calorimeter to provide a calibration liquid mixture whereby heat is generated, measuring the heat by the thermopiles and thereby providing a corresponding thermopile signal, and calibrating the chip-based microfluidic calorimeter by relating the thermopile signal to reference data of the deprotonating reaction.

Claims

1. A calibration method for calibrating a chip-based microfluidic calorimeter, wherein the chip-based microfluidic calorimeter comprises one or more thermopiles, wherein the calibration method uses the deprotonating reaction of a phosphate group, the method comprising: providing calibration liquids comprising (i) a buffer with a pH range of at least 7-9 and (ii) a first compound with a phosphate group which is protonated in a pH range of at least 3-6, and mixing these calibration liquids in the chip-based microfluidic calorimeter to provide a calibration liquid mixture whereby heat is generated, measuring the heat by the thermopiles and thereby providing a corresponding thermopile signal, and calibrating the chip-based microfluidic calorimeter by relating the thermopile signal to reference data of the deprotonating reaction.

2. The method according to claim 1, wherein the chip-based micro-fluidic calorimeter comprises a mixing chamber, wherein the mixing chamber has a mixing chamber length, and wherein the one or more thermopiles are configured to measure at different positions distributed over the mixing chamber length.

3. The method according to claim 2, wherein the mixing chamber has a volume selected from the range of 5-200 l.

4. The method according to claim 2, wherein after filling the mixing chamber with a volume equal to the volume of the mixing chamber with the calibration liquid mixture, flows of the calibration liquids to the mixing chamber is terminated and said heat is measured by said thermopiles.

5. The method according to claim 1, wherein the microfluidic calorimeter further comprises a mixing element, wherein the mixing element comprises one or more of a multi-lamination micromixer, a chaotic mixer, and a split-and-recombine mixer.

6. The method according to claim 1, comprising sequentially providing a series of calibration liquids having different concentrations of the first compound to the microfluidic calorimeter, measuring the heat by the thermopiles thereby providing corresponding thermopile signals, and calibrating the chip-based microfluidic calorimeter by relating the thermopile signals to reference data of the deprotonating reaction.

7. The method according to claim 1, wherein the method further comprises thermally equilibrating the calibration liquids prior to providing said calibration liquid mixture.

8. The method according to claim 1, wherein the reference data of the deprotonating reaction are based on isothermal titration calorimetry.

9. The method according to claim 8, wherein the method further comprises executing a further calibration method with the calibration liquids, wherein the further calibration method comprises isothermal titration calorimetry, for generating said reference data.

10. The method according to claim 1, wherein the reference data comprise kinetic reference data.

11. The method according to claim 1, wherein the phosphate group comprises phosphate (PO.sub.4.sup.3).

12. The method according to claim 1, wherein the buffer comprises 3-(N-morpholino)propanesulfonic acid (MOPS) and wherein the first compound comprises ATP.

13. A chip based microfluidic calorimeter calibrated according to the method according to claim 1.

14. Use of a chip-based microfluidic calorimeter according to claim 13, for measuring an enzymatic activity.

15. A calibration kit comprising a set calibration liquids comprising (i) a buffer with a pH range of at least 7-9, and (ii) a first compound with a phosphate group which is protonated in a pH range of at least 3-6, and optionally a manual for calibrating a chip based microfluidic calorimeter with the set of calibration liquids.

16. The calibration kit according to claim 15, comprising a first container comprising a first calibration liquid comprising said buffer, and comprising a plurality of second containers comprising said first compound, wherein each second container comprises a second calibration liquid with mutually different concentrations of said first compound.

17. The calibration kit according to claim 15, wherein the buffer comprises 3-(N-morpholino)propanesulfonic acid (MOPS) and wherein the first compound comprises ATP.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which:

[0041] FIG. 1a: Schematic overview of the ChipCal. Samples are loaded via a first inlet and a second inlet and simultaneously pumped through the microfluidic system: tubing, the heat exchanger and finally into the flow cell were the flow is stopped and changes in heat are registered by the four thermopiles; FIG. 1b schematically depicts a perspective view of an embodiment of the reaction chamber and thermopiles;

[0042] FIG. 2 schematically depicts some possible stages of the calibration method;

[0043] FIG. 3 schematically depicts a calibration kit;

[0044] FIGS. 4a-d show some data from calibration measurements;

[0045] FIGS. 5a-5d depict some measurements of the enthalpy of an enzymatic reaction.

[0046] The schematic drawings are not necessarily on scale.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0047] As indicated above, FIG. 1a schematically depicts an embodiment of the chip-based microfluidic calorimeter 100. Calibration liquids 120 are loaded via inlets 101. Here, two different calibration liquids 121 and 121 are applied, and introduced via a first inlet 101a and a second inlet 101b, respectively. One may e.g. include the buffer and the other may include the acid as defined above. The calibration liquids 120 are simultaneously pumped through the tubing (microfluidics), and an optional heat exchanger for thermally equilibrating the calibration liquids. The calibration liquids 120 are flowed into the mixing chamber (or reaction chamber), indicated with reference 130. Hereby, a calibration liquid mixture 123 is formed. The mixing chamber is indicated with dashed lines. When the mixing chamber 130 is filled the flow of the calibration liquids 120 may be stopped and changes in heat are registered by the (four) thermopiles indicated with reference 110. In an example, the volume of the mixing chamber or reaction chamber 130 is about 18 L.

[0048] In this embodiment, the mixing chamber 130 has a mixing chamber length L. The one or more thermopiles 110 are configured to measure (heat changes in the mixing chamber) at different positions 131 distributed over the mixing chamber length L. The thermopiles may not be in liquid contact with the calibration liquid mixture 123.

[0049] Reference 103 refers to a system fluid, which may be in liquid contact with the microfluidic system, which is indicated with reference 106. The system fluid may be used for flowing the cell. It can be used to inject the substrate and enzyme via the syringes into the cell. In embodiments, there might be an air gap between the system flow and the two components that are injected via the syringes. Here, by way of example the chip-based microfluidic calorimeter 100 also includes pumps 102, indicated with references 102a and 102b for the different channels for the different liquids, here the calibration liquids 121,122, respectively. Reference 104 indicates an outlet of the chip-based microfluidic calorimeter 100, such as to a waste reservoir. The rectangle may indicate a heat shield, substantially enclosing at least the mixing chamber and at least part of the thermopile(s) (at least the sensor part). Optionally, the heat shield may also enclose a thermal equilibration region 105, such as a heat exchanger. However, such a thermal equilibration region 105 may also be configured external of the heat shield. Further, the thermal equilibration region may include a heat exchanger and/or a heat sink, enclosing at least part of the micro-fluidic system 106.

[0050] For instance, in embodiments analogues to the device described by Lerchner et al. (see above), to ensure high signal resolution a calorimetric module is mounted inside a high-precision thermostat or heat shield which has a temperature stability of better than 100 K. The developed two-stage thermostat consists of two nested U-shaped frames. At the outer sides of the walls foil heaters are attached. To enable fast response the control temperature sensors (thermistors 10 k, BetaTherm) are placed inside the walls near the centre of the foil heaters. For temperature control two independent digital PID controllers with optimized parameters are used. The control temperatures for the outer and inner frame may be set to 25 and 25.3 C., respectively. A thermistor temperature sensor is placed inside the copper heat sink of the calorimetric module. The temperature is measured with a resolution of 6K and can be utilized for the correction of external temperature perturbations which are not completely suppressed by the thermostat. The inlets of the PMMA reaction chamber are connected with miniaturized piston pumps via Teflon tubes. The piston pumps (LEE LPV50) are part of fluid units and are operated together with sets of micro-valves (LEE LFVA) for reactant selection. Typical volume flow rates are ranged from 5 to 30 l/min. Volume flow rates higher than 50 l/min may exceed the capacity of the fluid heat exchanger. For the temperature equilibration of the liquids micro-machined heat exchangers (IPHT Jena) are used whose dead volumes are 15 l in each case. At first the liquid flows pass heat exchangers attached at the inner frame of the thermostat. A final temperature equilibration is achieved by heat exchangers attached at the bottom side of the copper heat sink plate. If volume increments of less than 15 l are injected optimal thermal adaptation of the reactants is assured. Further, the calorimetric system is equipped with an electronic unit for data acquisition, automatically operation of the fluid units and performing of the temperature control. The user interface is realized by a PC which is connected to the electronic unit.

[0051] FIG. 1b schematically depicts a perspective view of the mixing chamber 130, here by way of example a tubular mixing chamber. However, the mixing chamber may also have a square or rectangular cross-section. The mixing chamber is defined by a mixing chamber wall (and one or more inlets and an outlet). The mixing chamber wall may e.g. be made from Poly(methyl methacrylate) (PMMA), Polydimethylsiloxane (PDMS), etc., or other suitable polymers for micrcofluidic devices. At different positions 131 along the mixing chamber 130 thermopiles 110 are configured for measuring the heat generated within the mixing chamber 130.

[0052] FIG. 2 schematically depicts some possible stages of the calibration method, including a first stage I of providing the calibration liquids 120, an optional equilibration stage II, a mixing and measuring stage III wherein the mixing of the calibration liquids 121,122 provide the calibration liquid mixture 123 (and heat) and wherein the generated heat can be measured. This stage is followed by a calibration stage IV. Then, the calorimeter can be used for other measurements.

[0053] FIG. 3 schematically depicts an embodiment of a 15 calibration kit 200 comprising a set calibration liquids 120 comprising a buffer with a pH range of at least 7-9 (calibration liquid 121), and another calibration liquid 122, comprising a first compound which loses a proton at this pH, such as a phosphate group which is protonated in a pH range of at least 3-6. Optionally, the kit may further include manual 150 for calibrating a chip based micro-fluidic calorimeter 100 with the set of calibration liquids 120. Here, the kit especially includes a plurality of second containers 222 comprising said first compound, wherein each second container comprises a second calibration liquid 122 with mutually different concentrations of said first compound. Reference 221 refers to a first container, containing the first calibration liquid comprising the buffer. The term manual may refer to written information on one of these container, or a separte sheet, folder, booklet, or book, etc., with written information. However, the term manual may also refer to a manual on the internet. Hence, also a QR code, or other code, facilitate a user to go to an internet page with such manual is herein considered a manual.

EXAMPLES

[0054] All experiments were performed at 37 C. unless noted otherwise. The experiments were not performed at room temperature since this can change between days and between points of time in a day, and enthalpy changes can be influenced by environment temperature. It was specifically chosen to work at 37 Celsius since the enzymes used for this research performed better at this temperature.

[0055] MOPS, p-nitrophenyl phosphate, HCl and ATP were obtained from Sigma-Aldrich. KCl, and TRIS were obtained from Merck. NaCl, NaOH, KH.sub.2PO.sub.4 and MgCl.sub.2 were obtained from J. T. Baker. A second batch of ATP was acquired from Roche.

[0056] Alkaline phosphatase from Bovine intestinal mucosa was used, which was obtained from Sigma-Aldrich. The pH of the solutions was adjusted by using HCl or NaOH solutions in Milli-Q water.

[0057] The ChipCal instrument and software were provided by TTP LabTech. The samples were filtered and degassed prior to the experiments, to prevent clogging and the forming of extra air bubbles respectively. Enzymatic solutions, or other solutions also named samples, can e.g. be injected via a pen in the first inlet 101a pen into inlet 101a (FIG. 1). Substrate solutions, or other solutions, can e.g. be injected via a pen into second inlet 101b (FIG. 1). Between each experiment, a quick wash program was run to clean the flowcell with the system fluid. At the end of a set of experiments, a full wash program was performed with detergent (provided by TTP LabTech) and subsequently a clean water program was performed with Milli-Q water.

[0058] When a reaction occurs in the cell of the ChipCal, the thermopiles register a signal, taking five measuring points per second. However, there are also other phenomena that contribute to the signal, i.e. friction heat because of laminar flow and heat of dilution. These contributions can be independently measured by performing several blanks. The true signal, caused by the reaction, can be acquired by correcting for these blanks. First, when injecting two identical solutions in the flowcell, a change in temperature is observed by the thermopiles due to the physical forces of the two flows colliding and flowing through the cell. To determine this factor, one performs the experiment using the system fluid as both samples. Secondly, when a sample in solution is injected into the flowcell, the dilution of this compound into the other sample is also measurable as a change in temperature, the enthalpy of solution. To correct for this phenomenon, the dilution enthalpies for both samples may have to be determined. To determine this factor, one may perform the experiment of injecting one of the samples in one inlet and a sample solely containing the system fluid, the solvent, in the other inlet. By subtracting the friction blank from this signal acquired from this experiment, the change in heat registered by the thermopiles, caused by the dilution can be obtained. To determine the signal caused by the reaction, one may have to subtract the two dilutions blanks from the total signal. Since the enthalpy change caused by physical forces also occur in both of these two blanks, this factor is in fact subtracted twice. Therefore, one finally may have to add the physical blank once to the equation to determine the registered enthalpy caused by the reaction. This procedure is summarised in the equation shown below:

H.sub.reaction=H.sub.totalH.sub.dilution sample AH.sub.dilution sample B+H.sub.friction

[0059] For isothermal titration calorimetry (ITC), the Microcal VP-ITC from Malvern was used. The machine was set on high feedback mode with a reference power of 15 Cal/s. The stirrer rotated at 502 rpm. The filtering time of the machine was set on 2 seconds. To check reproducibility, two measurement injections were performed per run, preceded by a 2 L injection to get rid of a possible headspace of air in the syringe that occurs during the initial filling of the syringe. For each measurement, 3 L of substrate was injected from the syringe to the cell in three seconds. Adequate measuring time was used to allow the reactions to be completed and for the signal to return to the baseline. Samples were degassed and heated up to experimental temperature prior to all experiments. The same solvent was used to dissolve all compounds used for a set of experiments. As a blank, the enthalpy of solution for the sample in the syringe was determined by injecting 3 L of the sample in the cell, containing solely the diluent. All experiments were performed in triplicate.

TRIS-HCl Calibration

ChipCal

[0060] 20 mM, 15 mM, 10 mM or 5 mM solutions of hydrochloric acid (HCl) in Milli-Q water were mixed with a 200 mM tris(hydroxymethyl)aminomethane (TRIS) solution in Milli-Q water, pH 10.6 in the flowcell. In the ChipCal, the TRIS solution was injected via a pen in the first inlet 101a, the HCl was injected via a pen in the second inlet 101b. Milli-Q water was used as the system fluid. For the blank, a 200 mM TRIS solution in Milli-Q water was injected via needle A and Milli-Q water was injected via needle B. All blanks and experiments were performed in triplicate. Finally, these experiments were repeated on different days, up to a total of three times. The solutions were prepared fresh each day to analyse the reproducibility.

ITC

[0061] 200 mM TRIS in Milli-Q water, pH 10.6, was injected in the measuring cell of the VP-ICT. The syringe was filled with a 1 mM HCl in Milli-Q water solution. The experiments were performed in triplicate.

ATP-MOPS Calibration

ChipCal

[0062] 20 mM, 15 mM, 10 mM or 5 mM solutions of adenosine triphosphate, disodium salt (ATP), in a 200 mM MOPS, 20 mM Sodium Chloride (NaCl) solution at pH 5.0 in Milli-Q water were prepared. Lower reactant concentrations were chosen to reduce the effect of the ATP on the pH of the buffer. This was done since MOPS has low buffering capacity at pH 5.0. The ATP solutions were injected via a pen in the first inlet 101a. Via a pen in the second inlet 101b, a 200 mM MOPS, 20 mM NaCl solution at pH 8.0 was injected. The MOPS buffer at pH 5.0 was used as the system fluid. To obtain the heat due to physical friction, the MOPS solution at pH 5.0 was injected in both pens. For the dilution of ATP, the ATP solutions were each injected with the MOPS buffer at pH 5.0. To correct for the different ionisation states of the MOPS buffer, MOPS at pH 5.0 was injected together with MOPS pH 8.0. To check the consistency of the calibration with ATP, a batch from a different producer, here called ATPo, was acquired and these experiments were repeated. To investigate the influence of temperature on the instrument and the reactions, the temperature outside the cell was set on 28 C. and the experiments were repeated for the original batch of ATP, from now called ATPx. All blanks and experiments were performed in triplicate. The experiments with the first batch of ATP at 37 C. were repeated up to three times on different days with freshly made solutions to check reproducibility. The experiments with ATPo were performed on the same day, with the same buffers as one set of the ATPx experiments to ensure comparability.

ITC

[0063] A 200 mM MOPS, 20 mM NaCl buffer at the pH of 7.0 was injected into the cell. The pH of 7.0 is the end pH when mixing the MOPS buffer at pH 5.0 and pH 8.0 one to one. The syringe was filled with 10 mM of ATP in a 200 mM MOPS, 20 mM NaCl buffer at pH 5.0. This was done for both batches of ATP. The experiments were performed in triplicate. To determine the influence of temperature, the instrument temperature was set at 28 C. and the experiments with ATPx were repeated.

PO4-MOPS Calibration

ChipCal

[0064] 20 mM, 15 mM, 10 mM or 5 mM solutions of KH.sub.2PO.sub.4 in a 200 mM MOPS, 20 mM NaCl solution at pH 5.0 in Milli-Q water were prepared on different days to check reproducibility. The concentrations of phosphate were doubled after the first set to increase change in heat caused by the reaction. Since the enthalpy change is calculated to J/mol, the first set was still usable. Again, low reactant concentrations were chosen to reduce the impact on the pH of the buffers. The PO.sub.4 solutions were injected via a pen in the first inlet 101a. Via a pen in the second inlet 101b, a 200 mM MOPS, 20 mM NaCl solution at pH 8.0 was injected. The MOPS buffer at pH 5.0 was used as the system fluid. To obtain the heat due to physical friction, the MOPS solution at pH 5.0 was injected in both pens. For the dilution of KH.sub.2PO.sub.4, the phosphate solutions were each injected with the MOPS buffer at pH 5.0. To correct for the different ionisation states of the MOPS buffer, MOPS at pH 5.0 was injected with MOPS pH 8.0. All blanks and experiments were performed in triplicate.

ITC

[0065] A 200 mM MOPS, 20 mM NaCl buffer at the pH of 7.0 was injected into the cell. The pH of 7.0 is the end pH when mixing the MOPS buffer at pH 5.0 and pH 8.0 one to one. The syringe was filled with 10 mM of KH.sub.2PO.sub.4 in a 200 mM MOPS, 20 mM NaCl buffer at pH 5.0. The experiments were performed in triplicate.

PO.SUB.4.-TRIS Calibration

ChipCal

[0066] 40 mM, 30 mM, 20 mM or 10 mM solutions of KH.sub.2PO.sub.4 in a 200 mM TRIS, 20 mM NaCl solution at pH 5.0 in Milli-Q water were prepared on different days to check reproducibility. The PO.sub.4 solutions were injected via a pen in the first inlet 101a. Via a pen in the second inlet 101b, a 200 mM TRIS, 20 mM NaCl solution at pH 9.0 was injected. The TRIS buffer at pH 5.0 was used as the system fluid. To obtain the heat due to physical friction, the TRIS solution at pH 5.0 was injected in both pens. For the dilution of KH.sub.2PO.sub.4, the phosphate solutions were each injected in inlet 101a while TRIS buffer at pH 5 was injected in inlet 101b. To correct for the different ionisation states of the TRIS buffer, TRIS at pH 5.0 was injected together with TRIS pH 9.0. All blanks and experiments were performed in triplicate. Also, these experiments repeated up to three times on different days with freshly made solutions to check reproducibility. Since the 200 mM TRIS solution has a significantly low buffer capacity at pH 5.0, experimental errors can more easily be made while preparing the buffers at this pH. Therefore, to test the robustness of this method, the experiments were repeated for 20 mM KH.sub.2PO.sub.4 solution in TRIS at pH 4.5, 5.0 and 5.5. The method was repeated with these three samples, in triplicate.

ITC

[0067] A 200 mM TRIS, 20 mM NaCl buffer at the pH of 7.5 was injected into the cell. The pH of 7.5 is the end pH when mixing the TRIS buffer at pH 5.0 and pH 9.0 one to one. The syringe was filled with 10 mM of KH.sub.2PO.sub.4 in a 200 mM TRIS, 20 mM NaCl buffer at pH 5.0. The experiments were performed in triplicates.

Validation of the ATP Reaction: .SUP.31.P-NMR

[0068] To validate the products of the reaction with ATP, Phosphorus-31 NMR experiments were performed with an Agilent 400-MR NMR. ATP solutions in 200 mM MOPS, 20 mM NaCl pH 5.0 or pH 7.0 were diluted in 10% D.sub.2O and placed in 5 mm diameter NMR sample tubes. The instrument was calibrated with 85% H.sub.3PO.sub.4 as an external standard. The experiments were performed at room temperature and the instrument was operating at 161.98 MHz. The relaxation delay was 1.0 second, the acquisition time 0.8 seconds and the spectral width 249 ppm.

[0069] The ATP reaction was also validated with LC-MS.

Diffusion Dependance

[0070] To evaluate the impact of diffusion, several different salts were dissolved in Milli-Q water. These samples were injected in pen A while Milli-Q water was injected in pen B to investigate the signals for dilution. For the friction blank, Milli-Q water was injected in both pens. Solely the shape of the curves was studied for these experiments. Therefore the experiments were only needed to be performed in duplicates to check reproducibility. The dilution experiments were compared with the curves of the TRIS-HCl reactions. Since the protonation of TRIS is a reaction that can be considered as happening immediate, this reaction is considered to be dependent on the diffusion of the protons. The concentrations of the different compounds were chosen based on the maximum extension of the signal caused by the heat of dilution. It was attempted to acquire signals with similar extensions. For optimal comparison, the maximal signal amplitudes were normalized to 100%. The compounds and concentrations used for this experiment can be found in table 1.

TABLE-US-00001 TABLE 1 The compounds used for the diffusion experiment and the concentration used in mole per litre. Compound Concentration (M) HCl 0.005 KCl 1.00 NaCl 1.00 KH.sub.2PO.sub.4 1.00

[0071] To test the concentration dependence of the diffusion pattern, two more dilution experiments were performed with K H.sub.2PO.sub.4. For these experiments, the friction blank corrected signals for the dilution of 0.25 M and 1.0 M of K H.sub.2PO.sub.4 in Milli-Q water were again normalized and thereafter compared.

Enzyme Activity Measurements

[0072] Alkaline Phosphatase (AP) from bovine intestinal mucosa was used to convert para-nitrophenyl phosphate (PNPP) into para-nitrophenol (PNP) and phosphate. For inhibition studies, KH.sub.2PO.sub.4 was added to the reaction mixtures, up to an end concentration of 2 mM. All compounds were dissolved in a 200 mM TRIS, 20 mM NaCl buffer at pH 8.5. The pH optimum for AP is at pH 10, however, due to the fact that the ChipCal might be damaged by high pH, it was chosen to work at a more neutral pH.

[0073] First, the properties of alkaline phosphatase were obtained by determining the Michaelis Menten curve from activity measurements using UV visible spectrophotometry. The conversion of several concentrations of PNPP, between 5 and 400 by 0.0667 nM of AP in TRIS buffer at pH 8.8, was observed by UV-vis at wavelengths of 405 and 420 nm at 37 C. By monitoring the initial increase in absorption and using a calibration curve for PNP, the initial rates at different substrate concentrations were obtained. At 405 nm a molar extinction coefficient of 14,500 M.sup.1 cm.sup.1 was found.

[0074] In order to make the comparison between UV-vis and the ChipCal, the conversion of PNPP by alkaline phosphatase was recorded at various wavelengths. However, only the wavelengths of 475 nm, 477 nm and 480 nm were used to determine the PNP concentration over time. As stated before, the maximum absorbance of PNP lies at 405 nm, but at this wavelength, the instrument reaches its limitations too quickly. Therefore, wavelengths at the shoulder of the absorbance curves were used to determine the product formation. Glass cuvettes with a light path length of 1 cm were used. The solutions were stirred continuously by using a magnetic stirrer to ensure a homogeneous solution. AP was added at an end concentration of 10 nM to a solution of 5 mM PNPP, with or without 2 mM of PO.sub.4.

[0075] In the ChipCal, a 20 nM AP solution was injected by pen A and a 10 mM PNPP solution, with or without 4 mM PO.sub.4, was injected via needle B. Extra washing programs were performed between the experiments to ensure removal of any remaining enzyme.

[0076] To compare the rates of the enzyme over time measured by the two instruments, the rate of the enzyme was determined by converting the signals at specific times points. For converting the signals obtained from the ChipCal, in the aim to reduce the influence of the noise, the average of ten measured points around a time point were taken to get to a mean signal. These ten points span two seconds of measurements and should not be influenced by a significant change in rate of the enzyme. For the UV vis experiments, the slope of the obtained signal was determined at the set time points to determine the rate. These slopes were acquired by taking the derivative of twenty signal points around the set time points. These twenty points span twenty seconds of measurement and should not be influenced by a significant change in rate of the enzyme.

[0077] To obtain enthalpies (AH) of reactions the reactions were reproduced in a 1.4 ml isothermal titration calorimeter, the Microcal VP-ITC from Malvern. Using this instrument, the conditions present in the ChipCal flowcell were applied to obtain relevant reactions enthalpies per mole of substrate. The enthalpy values acquired by the ITC were then used to determine the properties of the thermopiles and the instrument. By using sufficient time for the reactions occurring to complete the integrals of the signals gained from the ChipCal and ITC, in V.Math.s per mole and in Joule per mole respectively, can be determined. By combining the values of these integrals, the thermopile sensitivity in Volts times seconds per Joule (V.Math.s/J), also noted as Volts per Watt (V/W), can be acquired.

[0078] The ChipCal is able to take up two samples of each 18 L with two injection pens. The samples are guided through the tubing of the instrument by the system fluid. To prevent diffusion of the samples into the system fluid, the samples are flanked by two air bubbles of 3 L (air gaps). The compounds for the samples should all be dissolved in the same diluent to decrease the background signal caused by the heat of dilution. For this reason, the system fluid, which is also used to flush through the tubing and the flow cell after an experiment, should be this solvent as well. This is necessary to decrease the dilution effects within the flow cell due to system fluid that may stick to the walls of the cell (Maskow, Schubert et al. 2011). Between experiments, the system can be programmed to flush the instrument using the software provided. The minimal diameter of the tubing within the system is as narrow as 0.4 mm and is thus susceptible to clogging. It is therefore advised to use water-soluble compounds and filter all samples prior to the experiments.

[0079] When the two samples of 18 L are taken up by the needles, they are pumped through the system by two individual pumps at a flow rate of around 75 L/minute. The samples are guided through a heat exchanger and finally brought together at the beginning of the measuring cell. The filling of this cell takes approximately 7 seconds. Thereafter, the flow is stopped and the heat measurement starts. The start is marked in the output by the instrument using a trigger.

[0080] The systems TRIS-HCl, MOPS-ATP and MOPS-KH.sub.2PO.sub.4.sup. were evaluated. The differences between the termopiles appears to be largest for the TRIS-HCL system. Comparing ATP and KH.sub.2PO.sub.4.sup., standard deviations for the ATP systems are smallest. Hence, for calibrating the calorimeter, ATP or KH.sub.2PO.sub.4.sup., may be suitable, with ATP providing the best results. Also a combination of TRIS and KH.sub.2PO.sub.4.sup., was tested but more reliable results are obtained with MOPS as buffer.

[0081] The calibration method is to calibrate chip-based microfluidic calorimeters. These type of calorimeters measure heat using thermopiles and the output of measurements is in micro volt, which should be covered to micro J/mol in order to be able to obtain rate of an enzymatic reaction. Hence, to do this, a calibration reaction with known enthalpy (micro J/mol) is required. The most widely used calibration method, which is based on the reaction of TRIS with HCl, is not suitable for chip-based microfluidic calorimeters because it does not account for diffusion. Herein, a new calibration method is proposed which is based on deprotonation of phosphate.

[0082] This method was applied to a chip-based microfluidic calorimeter for measuring enzymatic activity of alkaline phosphates. Using the new calibration method the activity measured by chip-based microfluidic calorimeter was similar to that obtained using UV-visible spectroscopy (FIG. 5).

[0083] In an embodiment, the steps used for calibration method and some relevant figures are described below: [0084] 1. Preparing MOPS buffer pH 8.0 [0085] 2. Preparing MOPS buffer pH 5, continuing different concentrations of phosphate or ATP, e.g. from 5 mM to 20 mM [0086] 3. Injecting the two buffers into the instrument. The two solutions will be mixed with a 1:1 ration in the microfluidic channel and the final pH will be around 7.0. As are result phosphate will be deprotonated and heat will be release, which will be recorded as a negative signal (micro volt) (FIGS. 4a and 4b). [0087] 4. Plotting the area under the curves recorded by the chip-based microfluidic calorimeter as a function of different concentrations of phosphate (FIGS. 4c and d), and obtaining the slope of a linear fit to the data (micro volts/mol) (FIGS. 4c and d). [0088] 6. Obtaining the enthalpy of deprotonation of phosphate in micro J/mol using a conventional calorimetric method such as isothermal titration calorimetry. [0089] 7. Calculating the sensitivity factor for thermopiles (volt/Watt) using the slope obtained in step 4 and the enthalpy of reaction. [0090] 8. The sensitivity factor for thermopiles can then be used to convert the data recorded for an enzymatic reaction to the rate of enzyme using the formula below:

Enzyme rate (mol/s)=Data recorded by calorimeter (micro volt)/[(sensitivity factor (volt/watt))(enthalpy of enzymatic reaction (micro J/mol)).

[0091] FIGS. 5a-d shows the application of our method to obtain rate of an enzyme called alkaline phosphatase with PNPP as substrate. FIG. 5a is the raw data recorded by a chip-based microfluidic calorimeter. FIG. 5b, the the raw data in FIG. 5a are converted to micro J/s using our calibration method. FIG. 5c, the data in FIG. 5a are used to obtain the rate of enzymatic reaction (micro J/s), and the results (line) are compared to those obtained using UV-visible spectroscopy (dots). FIG. 5d, is using the calibrated calorimeter to measure inhibition of alkaline phosphatase activity, with the upper curve indicating enzymatic activity in the absence of phosphate as inhibitor and with the lower curve indicating enzymatic activity in the presence of phosphate as inhibitor.

[0092] The term substantially herein, such as in substantially consists, will be understood by the person skilled in the art. The term substantially may also include embodiments with entirely, completely, all, etc. Hence, in embodiments the adjective substantially may also be removed. Where applicable, the term substantially may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. The term comprise includes also embodiments wherein the term comprises means consists of. The term and/or especially relates to one or more of the items mentioned before and after and/or. For instance, a phrase item 1 and/or item 2 and similar phrases may relate to one or more of item 1 and item 2. The term comprising may in an embodiment refer to consisting of but may in another embodiment also refer to containing at least the defined species and optionally one or more other species.

[0093] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0094] The devices herein are amongst others described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation or devices in operation.

[0095] It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. Use of the verb to comprise and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. The article a or an preceding an element does not exclude the presence of a plurality of such elements. The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In the device claim enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

[0096] The invention further applies to a device comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterizing features described in the description and/or shown in the attached drawings.

[0097] The various aspects discussed in this patent can be combined in order to provide additional advantages. Further, the person skilled in the art will understand that embodiments can be combined, and that also more than two embodiments can be combined. Furthermore, some of the features can form the basis for one or more divisional applications.