FIELD

[0001] Various example embodiments relate to temperature measurement using molecular temperature probes.

BACKGROUND

[0002] In biosensing, it is important to be able to monitor the temperature of the biosensors. Integrating thermal sensors into the biosensor may be cumbersome.

SUMMARY

[0003] According to some aspects, there is provided the subject-matter of the independent claims. Some example embodiments are defined in the dependent claims. The scope of protection sought for various example embodiments is set out by the independent claims. The example embodiments and features, if any, described in this specification that do not fall under the scope of the independent claims are to be interpreted as examples useful for understanding various example embodiments.

[0004] According to a first aspect, there is provided a micro fluidic device, comprising: a plurality of chambers comprising at least two different molecular temperature probes, wherein i) at least one of the plurality of chambers comprises, in a same chamber, a first molecular temperature probe and a second molecular temperature probe which is different than the first molecular temperature probe; or ii) a first chamber of the plurality of chambers comprises a first molecular temperature probe or a first mixture of probes; and a second chamber of the plurality of chambers comprises a second molecular temperature probe or a second mixture of probes, wherein the first molecular temperature probe is different than the second molecular temperature probe or the first mixture of probes is different than the second mixture of probes.

[0005] According to a second aspect, there is provided method for determining a temperature of a microfluidic device of any preceding claim; heating the microfluidic device to a first temperature; modifying the temperature of the microfluidic device by cyclic heating such that the temperature is caused to fluctuate above and below the first temperature; measuring a first fluorescence signal emitted by the first molecular temperature probe; measuring a second fluorescence signal emitted by the second molecular temperature probe, wherein the first fluorescence signal and the second fluorescence signal are temperature-dependent; calculating a derivative of the first fluorescence signal with respect to temperature; calculating a derivative of the second fluorescence signal with respect to temperature; determining the temperature of the microfluidic device based on the derivative of the first fluorescence signal and/or the derivative of the second fluorescence signal.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Fig. la shows, by way of example, molecular temperature probes;

[0007] Fig. lb shows, by way of a schematic example, how the structure of the probe changes in response to increasing temperature;

[0008] Fig. 1c shows dependency of fluorescence intensities of fluorophores of example molecular beacons on temperature;

[0009] Fig. 2 shows, by way of example, microfluidic devices;

[0010] Fig. 3 shows, by way of example, a flowchart of a method for determining a temperature of a microfluidic device;

[0011] Fig. 4 shows, by way of example, modulation cycles of temperature;

[0012] Fig. 5a shows, by way of example, fluorescence signals of two different molecular temperature probes;

[0013] Fig. 5b shows, by way of example, relative normalized derivatives of two different molecular temperature probes;

[0014] Fig. 6 shows, by way of example, a system for determining a temperature of a microfluidic device;

[0015] Fig. 7 shows, by way of example, measurement mechanisms with a single molecular temperature probe; and

[0016] Fig. 8 shows, by way of example, measurement mechanisms with two molecular temperature probes. DETAILED DESCRIPTION

[0017] In biosensing, it is important to be able to monitor the temperature of the biosensors. Exact temperature data is required for methods such as nucleic acid amplification to achieve the best performance. Molecular temperature probes may be used for accurate measurement of temperature on biosensors applying temperature-dependent fluorescence of the probes.

[0018] Fig. la shows, by way of example, molecular temperature probes, or molecular beacons (MBs). Molecular temperature probes 110, 120, 130 are hairpin-shaped molecules having unique temperature-dependent structural features. The probes have a stem sequence, i.e., arm sequence 115, and a loop sequence 116. At the ends of the arm sequences, the probe comprises a fluorophore molecule 112 and a quencher molecule 114. The fluorophore molecule may be referred to as a reporter molecule.

[0019] Fig. lb shows, by way of a schematic example, how the structure of the probe changes in response to increasing temperature. Denaturation temperature, i.e. melting or opening temperature, of the hairpin structure depends on the length of the stem and loop sequences. At lower temperatures, the hairpin structure 110, 120, 130 in a closed form, which means that the fluorophore 112 and the quencher 114 are close to each other, and no fluorescence is emitted. That is, the fluorescence is quenched due to the proximity of the quencher to the fluorophore. At higher temperatures, the hairpin structure is open, and fluorescence is active. That is, when the distance between the quencher and the fluorophore is large enough, the fluorescence is restored.

[0020] Examples of fluorophores 112, 122, 132 are 6-carboxyfluorescein (FAM), tetrachloro fluorescein (TET), Alexa® Fluor, ATTO, BODIPY®, Cascade Blue®, Cy® cyanine, EDANS, AMCA, DYOMICS, hexachloro-fluorescein (HEX), HiLyte™ Fluor, Marina Blue®, Oregon Green®, Pacific Blue®, Yakima Yellow®, Dragonfly Orange™, Texas Red®, rhodamine-6G, carboxy-X-rhodamine (ROX™), 5'-dichloro-dimethoxy- fluorescein (JOE), and 5-carboxytetramethylrhodamine (TAMRA).

[0021] Examples of quenchers 114, 124, 134 are N-[4-(4- dimethylamino)phenylazo]benzoic acid (DABCYL), Black Hole Quenchers (BHQ™, e.g., BHQ-1 and BHQ-2), DABCYL, DDQ, EDQ, TAMRA, QXL™ quenchers (e.g., QXL 490, QXL 570, QXL 670. ..), Iowa Black FW, and RQ, IRDye QC-1.

[0022] Let us consider example molecular beacons MB-47 FAM/BHQ-1, MB-64 FAM/BHQ-1 and MB-70 FAM/BHQ-1 and their fluorophores. Dependency of fluorescence intensities of these fluorophores on temperature is shown in Fig. 1c. The graph 160 corresponds to the intensity of the fluorophore of MB-47 FAM/BHQ-1, the graph 170 corresponds to the intensity of the fluorophore of MB-64 FAM/BHQ-1, and the graph 180 corresponds to the intensity of the fluorophore of MB-70 FAM/BHQ-1.

[0023] Fig. 2 shows, by way of examples, various designs of a microfluidic device 200, 201, 202, 203, 204, 205, 206, 207, e.g., a nucleic acid amplification chip. The microfluidic devices are used in various applications in medical diagnostics, biotechnology, etc.

[0024] In general, the micro fluidic device contains small scale structures (e.g., channels, sizes can vary between from a few micrometres to a few millimetres, typically several hundred micrometres) that are used to transport, mix, separate or process small volumes of liquid. The microfluidics are used in various fields, but they are very used in the nucleic acid analytic field. Nucleic acid analytics are utilized in point-of-care (PoC) diagnostics for example detection of pathogen from liquid biopsies (e.g., saliva, blood, water, etc.).

[0025] The used sample volumes are typically low, samples need different pretreatments and needed reagents or samples are expensive. With the same volume of reagent the sample may be analysed multiple times with microfluidics in comparison to conventional methods. In addition, the device may be multiplexed, that is, multiple analyses may be performed on a single sample, e.g., multiple different pathogens may be detected from the same sample.

[0026] With the microfluidics, diagnostics may be done under the field conditions or in resource-poor environment. However, nucleic acid amplification testing (NAAT) requires certain conditions to be fulfilled before being carried out. NAAT requires special enzymes (these will do the amplification), additives (e.g., buffering compounds that provide optimal environment), nucleotides (building blocks of nucleic acids), template (e.g., nucleic acids of the pathogen to be amplified), primers (short nucleic acid sequences that specify which portions of the template are amplified), detection reagents (e.g., nucleic acid stain) and aqueous solution to occur. In addition, heat is required for testing. The optimal temperature depends on the nucleic acid amplification techniques and used special enzymes. Without heat, the reaction cannot occur and typically changes in the heating will have huge impact to the final result. Uneven or non-stable heating can inhibit the amplification reaction leading to false result. Typically, microfluidic NAATs are heated chemically (e.g., handheld warmers) or in pre-calibrated heating devices (e.g., incubators). Conventional temperature sensors may be attached to these micro fluidic NAATs to monitor the temperature. However, these will increase the price and complexity of the test and still are not accurately keeping the temperature at desired range.

[0027] Microfluidics are made from various materials with varying physical properties and the compositions of these materials and designs for NAATs are almost unlimited. Measuring surroundings will affect how temperature is transferred from the surroundings to the amplification reaction solution. To measure the actual temperature that the amplification reaction undergoes, the used sensor must be in very close proximity or directly in the contact to the reaction chambers. The conventional temperature sensors may be too large in comparison with the micro fluidic chamber, and the size difference may cause inaccuracies. Use of sensors may also increase the price of the product and/or may cause problems in the mass manufacturing. In addition, additional components to receive and respond to the sensor feedback would be needed.

[0028] A pre-calibrated heating device still does not tell what the actual temperature inside reaction chambers is. The calibration is prone to human errors, and the heating device may need to be regularly maintained and calibrated, which will increase the end user prices. This will limit the possible end users (e.g., in case of home-testing). Depending on the used techniques, the temperature variations over two (>2) degrees Celsius (°C) have been considered to have significant effect on the nucleic acid amplification result.

[0029] In the exemplary micro fluidic devices 200, 201, 202, 203, 204, 205, 206, 207 of Fig. 2, the sample is put into one or more fluid inlets 300 to allow it to enter the microfluidics and special chambers. The chambers may be dead end chambers, for example. There it is processed, mixed, and contacted with other reagents to prepare for the nucleic acid amplification. The reaction chambers 210, 220, 230, 240 are either separated, but still in close vicinity of the molecular temperature probe chambers 250, 260, 270, 280, 290, or they have direct contact to each other’s via small channels. The reaction chambers may contain reagents for biochemical or biological reactions, such as enzyme-based nucleic acid amplification. When the conditions for the nucleic acid amplification reaction are fulfilled, the device is heated with a heating device (e.g., Peltier element or resistor) to start the reaction. For monitoring the reaction, the device is illuminated with the excitation source (e.g., laser LEDs) to enable emission by the fluorophores. The fluorescence is detected with the detector.

[0030] When temperature increases, the fluorescence of the selected molecular temperature probe starts to increase due to opening of the probe and decreased effect of the quenchers to the fluorophores and their fluorescence emission. However, the fluorescence intensity plateaus after large increase in the fluorescence, that is, the fluorescence is not going to increase infinitely with increasing temperature. This phenomenon follows the length and melting point temperatures of the used molecular temperature probes and thus the probes have their own characteristic fluorescence.

[0031] For example, a molecular temperature probe may be a single- stranded oligonucleotide-based bi-labelled fluorescent probe held in a hairpin-loop conformation by complementary stem sequences of oligonucleotides at both ends of the probe.

[0032] The molecular temperature probes have four parts: 1) relatively long oligonucleotide loops that are non-complementary, 2) stem formed by both termini’s short oligonucleotide sequences (5’- and 3 ’-ends) complimentary to each other, 3) fluorophore attached to the 5’ end and 4) quencher at 3’ end. This kind of beacon does not try to find amplified template. A template is a polynucleotide (ribonucleic acid, RNA, or deoxyribonucleic acid, DNA) that encodes the information from which another polynucleotide, of complementary sequence, is synthesized. This template may be used by the polymerases to form a new complementary sequence, which may be referred to as an amplicon or amplified product or amplified template. The molecular temperature probe has been designed in a way that it does not bind to a template or cannot find the template. The sequence of the molecular temperature probe is non-complementary to the template. When temperature decreases, these beacons will close, which will lead to the suppression of the fluorescence.

[0033] At the amplification chambers, the amplification is monitored by either utilizing other type of molecular beacons or nucleic acid stains. These molecular beacons have similar structure as the temperature molecular beacons described earlier, but their oligonucleotide sequence is designed in a way that it will be complimentary to the amplified template leading to hybridization. The duplex formed between the loop and the complementary amplified template is more stable than that of the stem because it involves more base pairs. This will lead to the separation of the fluorophore and quencher having similar result as the opening of temperature molecular beacon. Nucleic acid stains are not as specific as molecular beacons and they will have higher affinity towards doublestranded nucleic acids sequences (double-stranded DNA, dsDNA), but they have been shown to pose some affinity towards single-stranded nucleic acid sequences (singlestranded DNA, ssDNA, or ribonucleic acid (RNA)). In this way, we can monitor the fluorescence of both molecular beacons used for detection of amplification and molecular temperature probes used for detection of temperature at the same time.

[0034] For example, the microfluidic device 203, 204 comprises fluid inlet 300 and reaction chambers 210, 220 for biochemical or chemical components. The microfluidic device may comprise several fluid inlets 300. In some case, the micro fluidic device may also include outlet(s) 310 allowing fluid flowing out from the device. Choice of the components depends on the use case. Usually, liquid sample is applied into the inlet 300 and either using capillary or external pumps the fluid is directed to a single or multiple reaction chambers 210, 220, 230, 240, wherein biochemical reactions, such as nucleic acid amplification, and/or detection occur. For example, the microfluidic device may comprise at least one reaction chamber for biochemical or chemical components. The reaction chambers may be used for low- volume biochemical, chemical or biological reactions, such as enzyme-based nucleic acid amplification.

[0035] In addition, the microfluidic device 203, 204 comprises chambers 250, 260 for molecular temperature probes. Molecular temperature probes may be placed into chamber(s) separated from the actual sample reaction chambers, or the molecular temperature probes may be placed into the chambers that are connected to the reaction chambers via channels. The molecular probes may even be placed into the reaction chambers. However, the oligonucleotide sequences of the probes must be designed in a way that they do not interfere the reactions. For nucleic acid amplification reactions, the oligonucleotide sequence of the probe should not be complementary or similar to the amplified target or used template (e.g., DNA or RNA sequence) since the probe might cause false positive when it is amplified when the actual target is not present in the reaction. This is the case, if the amplification reaction is in contact with the molecular temperature probes. If they are not (e.g., they are in separate chambers), then it is not necessary to take this interference of amplification into account.

[0036] The probes may be re-hydrated with the sample matrix. Probes may be stored dehydrated since dehydrated probes may be stored for a longer time. Re-hydration may be performed using water content of the sample matrix, for example.

[0037] The microfluidic device 200, 201, 202, 203, 204, 205, 206, 207 comprises at least two different molecular temperature probes, that is, a first molecular temperature probe (a first probe) and a second molecular temperature probe (a second probe). For example, the probes may have different lengths and/or different nucleic acid sequences. Difference(s) in lengths and/or nucleic acid sequences cause different temperature dependency of the first probe and the second probe. The first probe may have different length than the second probe. The first probe may have a different nucleic acid sequence than the second probe. The first probe may have different length and a different nucleic acid sequence than the second probe.

[0038] More than two different probes enable measuring temperatures in a relative manner when the signal from two different probes are compared. In this case, the amplitude of temperature modulation, used to define the device temperature, can be unknown, because signal variation from two probes cancel each other. If one single probe is used, then the amplitude of the temperature modulation requires precise control. Usage of multiple probes enables also wider scale, since different probes are sensitive to different temperatures. For example, in a polymerase chain reaction (PCR), at least three different temperatures are used in different phases. Then, different pairs of probes may be used for different temperatures. For example, two pairs of probes may be used for each different temperature to enable control of the temperature such that the temperature may be kept at a suitable range. A first pair of probes may be used to monitor that the temperature does not decrease too much, and a second pair of probes may be used to monitor that the temperature does not increase too much.

[0039] The first probe and the second probe may be in the same chamber. The first probe is different from the first probe. The first probe and the second probe may form a mixture of probes. An example of a mixture of probes is a mixture comprising two different fluorophore-quencher pairs, such as MB-45 FAM/BHQ-1 and MB-47 FAM/BHQ-1. When the first probe and the second probe are in the same chamber, the first probe and the second probe are chosen such that the first probe is configured to emit fluorescence light of different colour than the second probe when exposed to excitation radiation.

[0040] Alternatively, the first probe, or a first mixture of probes, and the second probe, or a second mixture of probes, may be in different chambers. That is, a first chamber 250 may comprise a first probe or a first mixture of probes. A second chamber 260 may comprise a second probe or a second mixture of probes. When the first probe and the second probe are in different chambers, the first probe and the second probe may be chosen such that the first probe and the second probe are configured to emit fluorescence light of different colour or same colour when exposed to excitation radiation.

[0041] Number of the chambers on the micro fluidic device 200, 201, 202, 203, 204, 205, 206, 207 may be chosen according to the use case. Chambers 250, 260, 270, 280 and 290 may be located on different areas or zones on the microfluidic device. Thus, temperature may be monitored in various locations on the microfluidic device. The chambers may be interconnected in a multiple way.

[0042] The micro fluidic device may be heated by an integrated heating element or an external heating element. For example, the microfluidic device may be placed on a heating element, e.g., a Peltier element, or into an incubator or an oven. Alternatively, the microfluidic device may be heated using heating pads such as hand warmers, or physiologically between hands of a user, for example. The integrated heating element may comprise, for example, heating wires integrated into the microfluidic device.

[0043] Material of the micro fluidic device may be such that it has low autofluorescence in wavelengths of excitation radiation. Choice of the excitation radiation depends on the used fluorophore-quencher pair(s). For example, if the micro fluidic device is sensitive to wavelengths of blue light, autofluorescence of the device disturbs the detection of fluorescence of a certain probe in the wavelengths of blue light.

[0044] Materials of the microfluidic device may comprise e.g., polydimethylsiloxane (PDMS) and pressure sensitive adhesive (PSA).

[0045] Analytics and tests that are performed using the microfluidic device, such as nucleic acid analytics, require temperature handling. Typically, lab-on-chip technologies rely on external heating and temperature monitoring by external sensor heads. However, external sensor heads increase the cost and complexity of the test.

[0046] There is provided a method for monitoring the temperature inside the microfluidic device, for example, in the reaction chambers of the micro fluidics device.

[0047] Fig. 3 shows, by way of example, a flowchart of a method 300 for determining a temperature of a microfluidic device, such as any of the the microfluidic devices of Fig. 2.

[0048] The method comprises heating 310 the micro fluidic device to a first temperature. The first temperature is a target temperature and may be unknown. That is, it is not necessary to know the first temperature. The first temperature may be referred to as an average temperature value. The static fluorescence signals Ii, I2 emitted by the probes at the static first temperature may be measured. This static fluorescence signal values or strengths may be used in normalization of the derivatives, as described later below.

[0049] The method comprises modifying 320 the temperature of the microfluidic device by cyclic heating such that the temperature is caused to fluctuate above and below the first temperature. In other words, the temperature is modulated with varying amplitude and the amplitude of the signal is measured. For example, the amplitude above and below the first temperature may be e.g., 1 to 10 °C, e.g. 2, 3, 5, 10 °C. Suitable amplitude depends on the probes. Low amplitude may be preferable for nucleic acid amplification to avoid nonlinear behaviour.

[0050] Fig. 4 shows, by way of example, modulation cycles of temperature for a quantitative reverse transcription PCR (RT-qPCR) system. In this example, the first temperature 410 is approximately 40 °C. The modulation amplitude may increase along the increasing number of the cycles. In the example of Fig. 4, amplitudes of +/- 2, 3, 5 and 10 °C have been used, as clearly shown in the graph of Fig. 4. When the amplitude of approximately 10 °C has been used for some cycles, the micro fluidic device is heated to a second temperature 420. Thus, in addition to the modulation of temperature below and above some average value, the temperature of the microfluidic device may be gradually increased such that the average value of modulation increases in a stepped manner. The second temperature in this example is approximately 50 °C. The third temperature 430 is 60 °C, the fourth temperature 440 is 70 °C and the fifth temperature 450 is 80 °C. Using various different temperatures as average values, fluorescence of different probes that are sensitive to different temperatures may be activated. The first temperature 410, the second temperature 420, the third temperature 430, the fourth temperature 440 and the fifth temperature 450 may be referred to as temperature steps. The temperature is modulated or fluctuated below and above the temperature steps.

[0051] Duration of a fluctuation cycle may be e.g. 1 to 20 s, or 1 to 30 s. In nucleic acid amplification, it is preferable to achieve target temperatures in a fast manner enabling a good control on the biochemical reactions. Components used in nucleic acid amplification tolerate higher temperatures for short times, such as below 30 s. Molecular temperature probes react to the changes in temperature in a fast manner.

[0052] The probes, or the fluorophores, are excited by light, e.g. light emitting diode (LED). In response to excitation, the probes emit fluorescence signals. Intensities of the fluorescence signals are dependent on the temperature.

[0053] Referring back to Fig. 3, the method 300 comprises measuring 330 a first fluorescence signal emitted by the first molecular temperature probe and measuring 340 a second fluorescence signal emitted by the second molecular temperature probe. Fluorescence is read from hairpin structures using detector(s) suitable for that purpose. A detector may be a camera configured to detect emitted light from the excited fluorophores. Excitation filters and a diffuser may be positioned between the light source and the microfluidic device to filter out wavelengths overlapping with the fluorescence wavelength spectrum. Correspondingly, emission filters, e.g. colour filters, may be positioned between the microfluidic device and detector allowing only fluorescence light reaching the detector. The detector may be either cooled or non-cooled CMOS or CCD camera or alternately any photoactive device such as photodiode.

[0054] Fig. 5a shows, by way of example, fluorescence signals of two different molecular temperature probes. In this example, a first fluorescence signal 510 is the fluorescence signal of MB-70 FAM/BHQ-1 and a second fluorescence signal 520 is the fluorescence signal MB-64 FAM/BHQ-1. The amplitude modulation of the fluorescence signals is induced by the temperature modulation. The higher the modulation in temperature, the higher the change in fluorescence. The temperature has been modulated with amplitudes of +/- 2, 3, 5 and 10 °C, and the response of the fluorescence to the modulation is clearly shown in the graphs of Fig. 5a. [0055] Temperature of the micro fluidic device may be determined or calculated based on derivatives of the fluorescence signals with respect to temperature. During the temperature modulation, fluorescence intensity or intensities of the probes are monitored and fluorescence signal changes AIi and AI2 are measured. Derivatives AIi/AT and AI2/AT are then defined or calculated 350, 360 for the first and second probes, respectively. By dividing the derivatives with the static fluorescence intensity values, the normalized readings are obtained according to AI1/AT/I1 and AI2/AT/I2.

[0056] The method 300 comprises determining 370 the temperature of the microfluidic device based on the derivative of the first fluorescence signal and/or the derivative of the second fluorescence signal. If the accuracy of the temperature modulation is good, a single value, either AIi/AT/Ii or AI2/AT/I2, can be adequate to determine the temperature. Dependency between the temperatures and the intensities is known, and the temperature may be determined based on the known dependency. If AT is not accurate, the calculus continues by comparing two signals according to (AIi/AT/Ii)/(Al2/AT/l2) when AT is cancelled out and as a result the value (AIi/Ii)/(Al2/l2) is obtained to determine the temperature with an arbitrary modulation temperature span. The temperature span over which the derivative is calculated may be chosen to correspond to the modulation amplitudes, for example. In general, narrow temperature span may be accurate but noisier than a broader span.

[0057] Let us consider the fluorescence modulation below and above 30000000,000 a.u. in the example of Fig. 5a. The fluorescence modulation 532 is in response to modulation amplitude +/- 2°C. The fluorescence modulation 533 is in response to modulation amplitude +/- 3°C. The fluorescence modulation 535 is in response to modulation amplitude +/- 5°C. The fluorescence modulation 540 is in response to modulation amplitude +/- 10°C. Different derivatives may be calculated for these phases 532, 533, 535, 540, e.g., for each of these phases. For example, the derivative may be an average of derivatives of the cycles belonging to the same phase.

[0058] The derivatives are normalized. For example, the derivatives are divided by an average value, i.e., an average fluorescence signal strength. The average fluorescence signal strength corresponds to the fluorescence value around which, i.e., below and above which, the fluorescence fluctuates. The average fluorescence signal strength corresponds to the static measurement of the fluorescence signal before temperature modulation. For the phases 532, 533, 535, 540, the average fluorescence signal strength would be approximately 30000000,000 arbitrary units (a.u.). The normalized derivative of the first fluorescence signal is then AIi/AT/Ii and the normalized derivative of the second fluorescence signal is then AI2/AT/I2. Normalized derivatives are independent on the absolute value of the intensity.

[0059] The method comprises determining or calculating a ratio of the normalized derivatives. The ratio (AIi/AT/Ii)/(Al2/AT/l2) = (AIi/Ii)/(Al2/l2) corresponds to a certain temperature, and this correspondence is known for the used probes. That is, the dependency between different ratios and different temperatures is known. Temperature may be determined based on the known dependency between different ratios and temperatures.

[0060] Since the ratio of the normalized derivatives is used, there is no need to measure the absolute fluorescence value and the errors due to system variables are cancelled.

[0061] Let us summarize the determination of the temperature of the microfluidic device with the following example:

- measure static fluorescence signals after heating the device to the first temperature to obtain L of the first probe and I2 of the second probe

- temperature modulation around the first temperature and measurement of the fluorescence signals, e.g., amplitude of the signals, to obtain AL and AL

- calculate derivatives of the signals to obtain AL/AT and AI2/AT

- normalize the derivatives to obtain All/ AT/L and AI2/AT/I2

- If AT is accurate, determination of the temperature may be calculated from All/ AT/L or AI2/AT/I2

- If AT is not accurate enough, normalized derivatives from different probes are compared: (AII/AT/II)/(AI ₂ /AT/I ₂ ) reducing to (AII/II)/(AI ₂ /I ₂ )

[0062] Fig. 5b shows, by way of example, relative normalized derivatives calculated as ratios between two different molecular temperature probes. A plurality of samples and multiple amplitude modulation rounds were used to obtain the results or data points shown in Fig. 5b. Datasets show the obtained relative normalized values (All/ AT/Ii) / (AI2/AT/I2) when the temperature was modulated around a set value of 40°C, 50°C, 60°C, 70°C and 80°C. The set temperature modulation AT was either 2°C, 3°C, 5°C or 10°C. Four separate samples with repeated modulation rounds were measured to obtain statistical variation. As seen from a group of relative normalized derivatives 560 corresponding to temperature modulations below and above an average temperature the values converge with increasing modulation amplitude. With AT value of 2 °C the deviation was highest due to noise in the signal (AIi/ AT/Ii)/ (AI2/AT/I2). However, with increasing AT nonlinear effects in signal vs. temperature increase resulting in converged but imprecise value. Therefore, depending on the used probe molecules, modulation temperature needs to be optimized.

[0063] The method as disclosed herein enables temperature monitoring inside the chip, e.g., inside the reaction chambers, without need for external sensors or additional external components. Fluorescence is measured in any case, since the fluorescence of the samples in the reaction chambers is measured. The method as disclosed herein enables accurate temperature monitoring without need to acquire calibration curves. The method as disclosed herein enables inexpensive and reliable temperature monitoring for biosensing.

[0064] Fig. 6 shows, by way of example, a system for determining a temperature of a microfluidic device. The system comprises the microfluidic device 690, such as the microfluidic device 200, 201, 202, 203, 204, 205, 206, 207 of Fig. 2, comprising at least two different molecular temperature probes. The system comprises a heating element 691, such as a Peltier element, for heating the micro fluidic device 690. The heating element may be an integrated heating element or an external heating element, an incubator or an oven, for example. The system comprises a light source 692, for exciting the fluorescence molecules of the probes. For example, a LED may be used as the light source. A diffuser, lens, colour filter or other optical element 693 may be positioned between the light source 692 and the micro fluidic device 690. The system comprises a fluorescence measurement device 694, e.g., a detector, such as a CCD camera, for detecting the emitted fluorescence. A colour filter 695 may be positioned between the microfluidic device 690 and the detector 694.

[0065] The system comprises an apparatus 600 for analysing the measurement results and calculating the temperature based on the measurement results. In at least some embodiments, the apparatus 600 may be configured to control the heating element 691, for example, to perform cyclic heating. The apparatus 600 may be a computer such as a laptop, personal computer, or mobile tablet computer, for example. The apparatus comprises one or more processors 610 which may be means for performing method steps in the apparatus. The processor 610 may be configured to perform actions by computer readable instructions.

[0066] The apparatus 600 comprises at least one memory 620. Memory 620 may be at least in part accessible to processor 610. Memory 620 may be at least in part comprised in processor 610. Memory 620 may be means for storing information. Memory 620 may comprise or store computer instructions that processor 610 is configured to execute. When computer instructions configured to cause processor 610 to perform certain actions are stored in memory 620, and apparatus 600 overall is configured to run under the direction of processor 610 using computer instructions from memory 620, processor 610 may be considered to be configured to perform said certain actions. Memory 620 may be at least in part external to apparatus 600 but accessible to apparatus 600.

[0067] Apparatus comprises communication means such as a transmitter 630 and a receiver 640. Transmitter 630 and receiver 640 may be configured to transmit and receive, respectively, information wirelessly and/or via a wired connection. The apparatus 600 may receive 699 measurement results from the fluorescence measurement device 694 via a receiver 640. The receiver 640 may be configured to receive from the fluorescence measurement device 694, the measured intensities of the probes, which may be stored in one or more memories of the computer. The computer comprises computer readable instructions stored in a memory for analysing the measurement results and calculating the temperature according to the method as disclosed herein. The receiver may receive the measurements from the measurement device wirelessly or via a wired connection. Alternatively, the measurements may be transferred from the measurement device to the apparatus 600 using a memory stick, for example. Alternatively, the fluorescence measurement device may store the measurements into a cloud and the computer may retrieve the measurements from the cloud. After analysing the measurements and calculating the temperature, the computer may store the result to the memory, and/or transmit using a transmitter 630 the result to the cloud, and/or display the result to a user via a user interface, UI, 650. The apparatus 600 comprises or is connected to the UI 650. The UI 650 may comprise at least one of a display, a keyboard, a touchscreen, a mouse. A user may be able to operate apparatus 600 via UI 650. [0068] Fig. 7 shows, by way of examples, measurement mechanisms with a single molecular temperature probe. The microfluidic device 700, which may be the microfluidic device 203 of Fig. 2, comprises a single probe in a single chamber 701. The probe may be in the chamber 701 or chamber 702. The micro fluidic device 700 is heated 710, and the temperature probe activates, which is illustrated with the black circle 720. First, the Ii is measured and then the temperature is modulated 730. Then the AIi / AT is measured and AIi / AT / Ii is calculated. The system is controlled 740 or instructed to keep the current heat. If the accuracy of the temperature modulation is precise, the temperature of the microfluidic device may be determined based on this single value. Dependency between the temperatures and the intensities is known, and the temperature may be determined based on the known dependency.

[0069] Fig. 8 shows, by way of examples, measurement mechanisms with two molecular temperature probes. The microfluidic device 800, which may be the microfluidic device 203 of Fig. 2, comprises two different probes in two different chambers 801, 802. The first probe may be in the chamber 801 and the second probe may be in the chamber 802. The microfluidic device 800 is heated 810, and the temperature probes activate, which is illustrated with the black circle and the grey circle. First, the Ii and I2 are measured and then the temperature is modulated 820. Then the AIi / AT and AI2 / AT are measured and the AIi / AT / Ii and AI2 / AT / I2 are calculated. The system is controlled 830, 840 or instructed to keep the current heat. Finally, AIi / AT / Ii and AI2 / AT / I2 are compared 850 and the temperature difference AT is cancelled forming AIi / Ii and AI2 / I2. This ratio corresponds to a certain temperature, and this correspondence is known for the used probes. That is, the dependency between different ratios and different temperatures is known. Temperature may be determined based on the known dependency between different ratios and temperatures.

[0070] According to an example, a microfluidic device 203 comprises: a plurality of chambers comprising at least two different molecular temperature probes, wherein at least one chamber of the plurality of chamber is a reaction chamber 210 for biochemical or chemical components; wherein i) at least one of the plurality of chambers comprises, in a same chamber 250, a first molecular temperature probe 110 comprising arm sequences 115 and a loop sequence 116, and a fluorophore 112 and a quencher 114 at ends of the arm sequences; and a second molecular temperature probe 120 comprising arm sequences and a loop sequence, and a fluorophore 122 and a quencher 124 at ends of the arm sequences, wherein the second molecular temperature probe 120 has different length than the first molecular temperature probe 110; or ii) a first chamber 250 of the plurality of chambers comprises a first molecular temperature probe 110 comprising arm sequences 115 and a loop sequence 116, and a fluorophore 112 and a quencher 114 at ends of the arm sequences; and a second chamber 260 of the plurality of chambers comprises a second molecular temperature probe 120 comprising arm sequences and a loop sequence, and a fluorophore 122 and a quencher 124 at ends of the arm sequences, wherein the first molecular temperature probe 110 has different length than the second molecular temperature probe 120.