A METHOD, AN APPARATUS, AN ASSEMBLY AND A SYSTEM SUITABLE FOR DETERMINING A CHARACTERISTIC PROPERTY OF A MOLECULAR INTERACTION

Abstract

The invention concerns a method, an assembly and a system for determining a characteristic property of a molecular interaction. The method includes providing a liquid sample including a particle capable of being in a state of equilibrium and in a state of non-equilibrium. The particle includes a marker in at least one of its state of equilibrium and state of non-equilibrium. The method further includes bringing the particle in a state of non-equilibrium by subjecting the sample to a condition jump comprising a jump in temperature and/or pressure; reading out the marker as a function of time during at least a portion of a relaxation time for said particle, and determining said characteristic property of said molecular interaction.

Claims

1-111. (canceled)

112. A method for determining a characteristic property of a molecular interaction, the method comprising: providing a liquid sample comprising a particle capable of being in a state of equilibrium and in a state of non-equilibrium in the liquid sample, the particle comprises a marker in at least one of its state of equilibrium and state of non-equilibrium; bringing the particle in a state of non-equilibrium by subjecting the sample to a condition jump; reading out the marker as a function of time during at least a portion of a relaxation time for the particle; and determining the characteristic property of the molecular interaction, wherein the condition jump comprises subjecting the sample to a jump in temperature from at least one first temperature to a second condition at a second temperature and the method further comprises maintaining the second temperature during at least a part of the time of reading out of the marker.

113. The method of claim 112, wherein the reading out comprises performing two or more readings from different fractions of the sample.

114. The method of claim 112, wherein the particle being capable of being in a state of equilibrium and in a state of non-equilibrium: in that the sample comprises a binding partner for the particle, wherein the liquid sample comprises the particle and the binding partner in chemical equilibrium at the time of initiating the condition jump and wherein at least one of the particle or the binding partner comprises the marker; or in that the particle has a structure that depends on temperature, wherein the particle has a structure at equilibrium at the second condition, which differs from its structure prior to the condition jump.

115. The method of claim 112, wherein the particle is a protein and the structure difference and/or change is a difference and/or change in at least one folding of the protein.

116. The method of claim 112, wherein the particle has a conformation at equilibrium at the second condition, which differs from its conformation prior to the condition jump.

117. The method of claim 112, wherein the jump in temperature of the sample is performed in the microfluidic unit, the method comprises introducing the sample into the microfluidic unit, wherein the microfluidic unit is at least partly located in a temperature controlled maintaining compartment.

118. The method of claim 117, wherein the microfluidic unit comprises an introduction section to which the sample is introduced, the introduction section comprises a cross-sectional dimension of about 1 mm or less.

119. The method of claim 117, wherein the temperature controlled maintaining compartment is maintained at the second temperature during at least a portion of the relaxation time.

120. The method of claim 117, wherein the temperature controlled maintaining compartment is temperature controlled: by a method comprising blowing of air; by a method comprising fully or partly filling the compartment with liquid and/or vapor; or by a method comprising applying a high voltage to the sample while the sample is located in a container, which forms part of or comprises at least a part of the microfluidic unit.

121. The method of claim 112, wherein the temperature jump from the at least one first temperature to the second temperature comprises providing a temperature jump of at least about 2° C.

122. The method of claim 112, wherein the second temperature is from about 5° C. to about 50° C.

123. The method of claim 112, wherein the microfluidic unit comprises an introduction section and a reading out section, and wherein the reading out comprises performing readings of the sample while the sample is flowing in the microfluidic unit, wherein the reading out as a function of time comprises performing the two or more readings from different fractions of the sample as the sample is flowing in the reading section of the microfluidic unit.

124. The method of claim 112, wherein the reading out as a function of time comprises performing consecutive readings from different fractions of the sample as the respective sample fractions are passing a reading location of the microfluidic unit.

125. The method of claim 112, wherein the method comprises determining at least one of a kinetic parameter, a partitioning parameter, a degradation parameter, an oligomerization parameter, and a folding parameter.

126. The method of claim 112, wherein the molecular interaction comprises a liquid-liquid phase separation, wherein the particle comprises at least two different molecules and an optional additional solvent, which molecules are capable of forming a liquid-liquid phase separation at the condition prior to or after the temperature jump.

127. The method of claim 126, wherein the liquid sample at the time immediately prior to subjecting the sample to the temperature jump is in a single phase condition, and wherein the liquid-liquid phase separation comprises at least local formation of a first liquid phase with an interface to a second liquid phase.

128. The method of claim 127, wherein the first liquid phase and the seconds liquid phase differs from each other with respect to concentration and/or presence of at least one molecule.

129. The method of claim 127, wherein the temperature jump is a jump from a higher temperature to a lower temperature, wherein the sample is in a single-phase condition at the higher temperature.

130. The method of claim 126, wherein the sample is subjected to the temperature jump in the channel of the microfluidic unit and the reading out is performed in the channel, wherein the sample is fed to the channel at a pressure to ensure a selected velocity of the sample in the channel, wherein the velocity is adjustable.

131. An apparatus suitable for determining a characteristic property of a molecular interaction by the method of claim 112, the apparatus comprising: a sample compartment for containing at least one liquid mother sample; a withdrawing arrangement arranged for withdrawing a sample from a at least one mother sample stored in the sample compartment a condition jump arrangement arranged for performing a temperature jump of the sample from at least one first temperature to a second temperature, and at least one reader arrangement for reading at least one marker as a function of time, wherein the apparatus further comprises a temperature controlled maintaining compartment for maintaining the sample at the second condition during the reading out of the marker.

132. The apparatus of claim 131, wherein the maintaining compartment comprises a temperature controller arrangement comprising a blower for blowing air at a selected temperature and/or a liquid sprinkler for sprinkling liquid at a selected temperature and/or a liquid filler for fully or partly filling the maintaining compartment with liquid at a selected temperature.

133. An apparatus assembly, comprising the apparatus of claim 131 in combination with the microfluidic unit, wherein the microfluidic unit is at least partly located in the temperature controlled maintaining compartment.

Description

BRIEF DESCRIPTION OF THE EXAMPLES AND DRAWING

[0269] The invention is being illustrated further below in connection with examples and embodiments and with reference to the figures. The figures are schematic and may not be drawn to scale. The examples and embodiments are merely given to illustrate the invention and should not be interpreted to limit the scope of the invention

[0270] FIG. 1 illustrates an embodiment of a system of the invention comprising a computer system and an assembly of an apparatus and a microfluidic unit.

[0271] FIG. 2 illustrates a variation of the embodiment in FIG. 1.

[0272] FIGS. 3a-3e show examples of microfluidic units suitable for use in embodiments of the apparatus of the invention.

[0273] FIGS. 4a and 4b are diagrams showing a fluorescence intensity as a function of time as described in example 1.

[0274] FIGS. 5a-5g are diagrams showing a fluorescence intensity as a function of time as described in examples 2a-2g.

[0275] The system of FIG. 1 comprises an apparatus 1 suitable for determining a characteristic property of a molecular interaction and a microfluidic unit 4. The apparatus comprises a maintaining compartment 2 and a sample compartment 3 separated by a separating wall 14 having a passage for the microfluidic unit 4.

[0276] The sample compartment 3 comprises a plurality of mother sample chambers 7, arranged in a support unit 7a. The support unit 7a advantageously comprises a temperature controller for temperature controlling of mother samples in the respective mother sample chambers 7 to a selectable temperature. The sample compartment 3 comprises a withdrawing arrangement comprising a pump arrangement 5, connected to a plurality of withdrawing tubes 6. Each tube advantageously comprises a needle adapted for penetrating a cover membrane on the respective of mother sample chambers 7. The respective tubes 6 may be manually inserted into desired mother sample chambers, by penetrating the membrane of the mother sample chamber with the needles at their ends. In an embodiment, the apparatus 1 comprises a robot arm adapted for insert the tube(s) 6 into selected mother sample chamber(s).

[0277] In a variation of this embodiment the withdrawing arrangement comprising a single withdrawing tube.

[0278] The apparatus 1 comprises a hinged 1b lid 1a into the sample compartment 3 for providing access there to.

[0279] In this embodiment, the microfluidic unit 4 is a tube with a narrow diameter e.g. as described above. The tube 4 is connected to the pump arrangement, such that the pump can pump withdrawn mother sample into the microfluidic unit 4 at a desired pressure difference.

[0280] The maintaining compartment 2 comprises a computer unit 9 adapted for controlling the elements of the apparatus 1. The computer 9 is connected to a reader arrangement 11.

[0281] The maintaining compartment 2 comprises a condition jump arrangement 8, adapted for performing the temperature jump by conduction and/or convection e.g. as described above. The condition jump arrangement 8 may for example comprise a blower or a peltier element. A temperature controller arrangement 8a is connected with the condition jump arrangement 8, such that the temperature controller arrangement 8a may control the operation of the condition jump arrangement 8 and the temperature in the maintaining compartment 2.

[0282] A waist chamber 10 is located for collect used sample and optional cleaning fluid passed through the microfluidic unit 4

[0283] The microfluidic unit 4 has an introduction section 4a which is arranged adjacent to the condition jump arrangement 8. The microfluidic unit 4 also has a reading out section 4b, which is this embodiment is a single location at the microfluidic unit.

[0284] In use, the sample is withdrawn from one or more selected mother sample containers 7 by the tube(s) 6 and the pump arrangement 5 of the withdrawing arrangement.

[0285] The sample is fed into the microfluidic unit 4 into the introduction section 4a at a relatively high pressure difference to ensure that the introduction of sample is performed relatively fast. When the sample has reach the introduction section 4a, the pump arrangement, the pressure provided by the pump arrangement 5 is reduced or fully stopped. In the introduction section 4a the condition jump arrangement 8 is heating the sample very fast to ensure a desired temperature jump.

[0286] Thereafter, pump arrangement 5 is pumping the sample to reach the read out section 4b. The pressure is reduced to provide that the sample is passing the read out section 4b at a desired slow velocity to ensure a desired long reading timed. While the sample is passing the read out section 4b, the reader arrangement 11 is performing a plurality readings at a desired reading rate e.g. as described above.

[0287] The variation of the system shown in FIG. 2 comprises a personal computer 12, with a screen 12a. The personal computer 12 is in data connection with the computer 9, incorporated in the apparatus 1. The computer system comprises the personal computer 12 and the computer 9.

[0288] FIG. 3a shows an embodiment of a suitable microfluidic unit in the form a long, substantially straight tube with a narrow inner diameter.

[0289] FIG. 3b shows an embodiment of a suitable microfluidic unit in the form a long, coiled tube with a narrow inner diameter.

[0290] FIG. 3c shows an embodiment of a suitable microfluidic unit in the form a microfluidic device 21, with a flat chamber 22 and an inlet 23 to the chamber 22.

[0291] FIG. 3d shows an embodiment of a suitable microfluidic unit in the form a microfluidic device 28, with a long coiled channel 29a. The channel has an inlet 29c, leading to an introduction section 29d, where a sample may be subjected to a temperature jump. The channel has a reading out section 29b.

[0292] FIG. 3e shows an embodiment of a suitable microfluidic unit in the form a chamber provided by crystallized aluminum oxide 24 with a membrane cover 25 and bottom. The sample may be introduced into the chamber via a tube 26. The figure also illustrates a part of the condition jump arrangement adapted for performing a pressure jump. The condition jump arrangement comprises a piezoelectric crystal stack 27 and a holding arm 27a adapted to hold the piezoelectric crystal stack 27 against the membrane 25.

Example 1—HSA-Fluorescein Binding Partner Assay

[0293] A sample comprising a molar concentration of human serum albumin (HSA) of 83 micro mol and a molar concentration of 10 nano mol of a binding partner to the HSA, namely Flourescein (fl) in a buffered solution at a pH value of 7.4.

[0294] An assay was performed as describe in connection with FIG. 1, where the temperature jump was a 10 degrees jump from 5° C. to 15° C. The resulting readings were plotted and are shown in FIG. 4a.

[0295] Another assay was performed as describe in connection with FIG. 1, where the temperature jump was a 20 degrees jump from 5° C. to 25° C. The resulting readings were plotted and is shown in FIG. 4b.

[0296] In FIG. 4a, the final temperature is 15° C. and the relaxation to equilibrium is governed by the rate constants at 15° C. In FIG. 4b, the final temperature is 25° C. and the relaxation to equilibrium is governed by the rate constants at 25° C. Kinetic rate constants are higher at higher temperatures compared to lower temperatures. Relaxation kinetics can be described by the relaxation time denoted by tau:

S=a+b(1−exp(−t/tau))

[0297] S is the signal obtained from the reader (in this case a fluorescence reader), a is a constant describing detection offset and or background, b is the magnitude of the change in signal between initial state and final state and it is time.

[0298] Tau is quantified by and appropriate fit to the data. In a more advanced data analysis, the relaxation may be modeled using several tau values is several relaxation processes are in play.

[0299] Tau is linked to the rate constants pertaining to the molecular property under study. For example, a 1-1 non-covalent interaction (A+I=AI) in which A is in large excess of I may be linked to tau according to:

Tau=1/(kon[A]+koff)

[0300] In which kon and koff are the rate constants pertaining to formation and dissociation of the complex AI.

Example 2a—LLPS Assay

[0301] A mother sample (a) was prepared.

[0302] The following materials were used in this or in the following examples:

[0303] FI-dextran: A fluorescently labeled dextran having a molar weight of about 7000 Dalton.

[0304] Dextran: A non-labeled dextran having a molar weight of about 200000 Dalton.

[0305] PEG: Poly(ethylene glycol), molar weight of about 6000 Dalton.

[0306] Water: Pure water (type II).

[0307] FI-HSA: A fluorescently labeled Human Serum Albumin.

[0308] An aqueous mother sample (a) were prepared from water, PEG and fl-dextran to have a concentration of PEG of 5 massl % and a concentration of fl-dextran of 20 nM.

[0309] An assay was performed as describe in connection with FIG. 1.

[0310] The prepared mother sample (a) was applied in a sample chamber 7 of the sample compartment 3 and the temperature of the mother sample was set to 50° C. The sample was withdrawn from the mother sample (a) and pumped into the introduction section of the tube in the maintaining compartment, where it was subjected to a 25 degrees temperature jump from 50° C. to 25° C. Fluorescent intensity readings were performed at the read out section as the sample passes through.

[0311] The resulting readings at the read out section are shown in FIG. 5a.

[0312] The reference “s” indicates the start of reading. The first few seconds of the readings, the sample has not fully reached the read out section. As the sample reaches the read out section, the signal raises to its maximal level and remains substantially stably during the remaining reading time until data end (DE). From this, it can be concluded that there remains one single phase from start to end of experiment. I.e. no liquid-liquid phase separation takes place.

Example 2b—LLPS Assay

[0313] A mother sample (b) was prepared from the same materials as listed in example 2a.

[0314] The aqueous mother sample (b) were prepared from water, Dextran, PEG and fl-dextran to have a concentration of PEG of 5 mass %, a concentration of Dextran of 1 mass % and a concentration of fl-dextran of 20 nM.

[0315] The assay was performed as described in example 2a.

[0316] The resulting readings at the read out section are shown in FIG. 5b.

[0317] The curve obtained in 5b is very similar to the curve of FIG. 5a, however, with a little instability immediately after having reached its maximal level as indicated with ref. 32.

[0318] In addition the maximal level reached in FIG. 5b, is slightly lower than the level reached in FIG. 5a.

[0319] These characteristic indicates that the single phase of the sample becomes instable and indicates signs of liquid-liquid phase separation e.g. formations of sprinkles or bobbles of a separated phase.

Example 2c—LLPS Assay

[0320] A mother sample (c) was prepared from the same materials as listed in example 2a.

[0321] The aqueous mother sample (c) were prepared from water, Dextran, PEG and fl-dextran to have a concentration of PEG of 5 mass %, a concentration of Dextran of 2 mass % and a concentration of fl-dextran of 20 nM.

[0322] The assay was performed as described in example 2a.

[0323] The resulting readings at the read out section are shown in FIG. 5c.

[0324] In the curve obtained in 5c a clear spike is visible immediately after the signal has reached its maximal level as indicated with ref. 33a. After the spike 33a the signal intensity drops to a lower level 33b, which level is also lower than the general max intensity level shown in FIGS. 5a and 5b.

[0325] These characteristic indicates that the sample has initiated liquid-liquid phase separation. The instability of the signal intensity at the lower level 33b also indicates formations of sprinkles or bobbles of a separated phase.

Example 2d—LLPS Assay

[0326] A mother sample (d) was prepared from the same materials as listed in example 2a.

[0327] The aqueous mother sample (d) were prepared from water, Dextran, PEG and fl-dextran to have a concentration of PEG of 5 mass %, a concentration of Dextran of 3 mass % and a concentration of fl-dextran of 20 nM.

[0328] The assay was performed as described in example 2a.

[0329] The resulting readings at the read out section are shown in FIG. 5d.

[0330] The curve obtained in 5d shows a very significant spike 34a and an increased instability of the intensity level 34b after the spike 34a.

[0331] In addition, it can be observed that the intensity level after the spike 34a is generally lower than in the previous LLPS assays with lower amount of Dextran.

[0332] These characteristic indicates a clear liquid-liquid phase separation of the sample and that formations of sprinkles or bobbles of a separated phase has taken place.

Example 2e—LLPS Assay

[0333] A mother sample (e) was prepared from the same materials as listed in example 2a.

[0334] The aqueous mother sample (e) were prepared from water, Dextran, PEG and fl-dextran to have a concentration of PEG of 5 mass %, a concentration of Dextran of 4 mass % and a concentration of fl-dextran of 20 nM.

[0335] The assay was performed as described in example 2a.

[0336] The resulting readings at the read out section are shown in FIG. 5e.

[0337] The curve obtained in 5e shows a very significant spike 35a. In addition, the intensity level 35b after the spike 35a is significantly lower than in the previous LLPS assays with lower amount of Dextran e.g. as in example 2d/FIG. 5d. Comparing the intensity level 35b after the spike 35a of FIG. 5e with the intensity level 34b after the spike 34a of FIG. 2d, the intensity level in 5e in almost 30% lower.

[0338] These characteristic indicates that the formations of sprinkles or bobbles of separated phase is larger in example 2e than in example 2d.

Example 2f—LLPS Assay

[0339] A mother sample (f) was prepared from the same materials as listed in example 2a.

[0340] The aqueous mother sample (f) were prepared from water, Dextran, PEG and fl-dextran to have a concentration of PEG of 5 mass %, a concentration of Dextran of 5 mass % and a concentration of fl-dextran of 20 nM.

[0341] The assay was performed as described in example 2a.

[0342] The resulting readings at the read out section are shown in FIG. 5f.

[0343] The curve obtained in 5f shows a very significant spike 36a. In addition, the intensity level 36b after the spike 35a is even lover lower than in example 2e/FIG. 5e. This indicates that the liquid-liquid phase separation is even more pregnant and that larger volume of sprinkles or bobbles of separated phase have been formed.

Example 2g—LLPS Assay

[0344] A mother sample (g) was prepared from the same materials as listed in example 2a.

[0345] The aqueous mother sample (f) were prepared from water, Dextran, PEG and fl-HSA to have a concentration of PEG of 5 mass %, a concentration of Dextran of 4 mass % and a concentration of fl-dextran of 50 nM.

[0346] The assay was performed as described in example 2a.

[0347] The resulting readings at the read out section are shown in FIG. 5g.

[0348] The curve obtained in 5g shows a very high and significant spike 37, clearly indicating the liquid-liquid phase separation takes place after a few minutes from the temperature jump. After the spike 37, the intensity level drops about 45% and the intensity signal shows increasingly instability over time, which is a clear indication of formations of sprinkles or bobbles of separated phase.